Pain treatment using ERK2 inhibitors

ABSTRACT

This application describes methods and compositions for reducing, inhibiting and/or treating pain that involve use of ERK2 inhibitors.

This application is a divisional of U.S. application Ser. No. 12/997,836, filed Dec. 13, 2010, now U.S. Pat. No. 8,951,979, to be issued on Feb. 10, 2015, which is national stage application under 35 U.S.C. §371 of PCT/US2009/003523, filed Jun. 12, 2009, Published as WO 2009/151620 and published on Dec. 17, 2009, and which applications claim benefit of the filing date of U.S. Provisional Ser. No. 61/061,254, filed Jun. 13, 2008, the contents of which applications are specifically incorporated herein by reference.

This invention was made with government support from the National Institute on Drug Abuse (NIDA) grant numbers DA001457 and DA000198 (CEI), NIDA training grant DA007274 and NIDA center grant DA005130. The U.S. government has certain rights in this invention.

BACKGROUND OF THE INVENTION

A variety of treatments have been proposed and evaluated for the treatment of pain, including medications, acupuncture, local electrical stimulation, brain stimulation, and surgery. Psychotherapy, relaxation therapy, biofeedback, and behavior modification have also been employed in attempts to treat pain. Despite the many proposed therapies, pain remains an important and increasingly common medical complaint. Moreover, the root causes of pain are sometimes difficult to determine, and frequently are difficult to treat and control.

SUMMARY OF THE INVENTION

This application describes compositions and methods for selectively inhibiting extracellular signal-regulated kinase 2 (ERK2) that are useful for treating pain. Experiments described herein demonstrate that inhibition of ERK2 by use of an ERK2 siRNA delivered by a neurotropic adeno-associated viral vector reduces pain sensitivity in adult mice. Mice were injected in their hind paws with Complete Freund's adjuvant (CFA) to induce peripheral inflammation, mechanical allodynia and thermal hyperalgesia that persisted for at least 96 hours. The ERK2 siRNA protected the animals from developing mechanical allodynia and thermal hyperalgesia throughout the 96 hours after CFA. These findings indicate that ERK2 is involved in the development of pain hypersensitivity and that inhibition of ERK2 expression or activity can reduce or inhibit pain and the development of pain.

One aspect of the invention is a method for treating or inhibiting pain in an animal comprising administering to the animal an inhibitor of extracellular signal-regulated kinase 2 (ERK2) to thereby treat or inhibit pain in the animal. The pain that is treated or inhibited can be chronic pain, acute pain, inflammatory pain, somatic pain, visceral pain, neuropathic pain, and combinations thereof. In some embodiments, the pain that is treated is inflammatory pain. In other embodiments, the pain that is treated is somatic pain or visceral pain. In further embodiments, the origin of pain that is treated is unknown or arises from a combination of causes or pain types. The animal treated can be a human, domesticated animal, experimental animal or a zoo animal. In some embodiments, the inhibitor is administered locally. In other embodiments, the inhibitor is administered systemically (e.g., orally or parenterally). The inhibitor can be any ERK2 inhibitor, for example, the inhibitor can be an antibody, a nucleic acid that inhibits the expression of ERK2, a compound (e.g., a small molecule), as well as other types of ERK2 inhibitors and combinations thereof.

Thus, in some embodiments, the inhibitor is an anti-ERK2 antibody that specifically binds to ERK2. Such an anti-ERK2 antibody can be combined with other ERK2 inhibitors and/or pain medications.

In other embodiments, the inhibitor is a nucleic acid that can inhibit the expression of ERK2. Such a nucleic acid can, for example, hybridize to an mRNA encoding a ERK2 polypeptide with SEQ ID NO: 2. In some embodiments, the nucleic acid can hybridize to an ERK2 polynucleotide comprising SEQ ID NO:2 or SEQ ID NO:771. Examples of inhibitory nucleic acids that can be used in the methods and compositions described herein include antisense nucleic acids, small interfering RNA, ribozyme nucleic acids and combinations thereof. Such inhibitory nucleic acids can have a modified backbone or one or more non-natural internucleoside linkages.

Thus, the nucleic acid can, for example, be a small interfering RNA comprising a DNA or RNA sequence with any of SEQ ID NO:3-162, 166-764, or a combination thereof; or a DNA or RNA that can specifically hybridize to any of SEQ ID NO:3-162, 166-764, or a combination thereof. Such a nucleic acid can be a small interfering RNA comprising a DNA or RNA sequence with any of SEQ ID NO:773-775, or a combination thereof; or a DNA or RNA that can specifically hybridize to any of SEQ ID NO:773-775, or a combination thereof. In some embodiments, the nucleic acid is a small interfering RNA comprising a DNA or RNA sequence corresponding to any one SEQ ID NO:779, 782, 785, or a combination thereof; or a DNA or RNA that can specifically hybridize to any one SEQ ID NO:779, 782, 785, or a combination thereof.

The nucleic acid can be encoded within an expression cassette comprising a promoter and a polynucleotide segment comprising a DNA or RNA corresponding to any of SEQ ID NO: SEQ ID NO:3-162, 166-764, 773-775, 779, 782, 785 or a combination thereof. Such an expression cassette can also comprise a promoter and a polynucleotide segment comprising a DNA or RNA that can hybridize to any of SEQ ID NO: SEQ ID NO:3-162, 166-764, 773-775, 779, 782, 785 or a combination thereof.

Thus, for example, the segment can have the sequence X-L-Y, wherein X is a sense sequence, L is a spacer linked to the 3′ end of the sense sequence, and Y is an antisense sequence linked to the 3′ end of the linker, and wherein the Y antisense sequence is complementary to the X sequence so that upon expression of the polynucleotide segment, a short hairpin RNA (shRNA) is generated.

The expression cassette can be present in an expression vector where such an expression vector can be a viral vector. Examples of viral vectors that can be used in the methods and compositions of the invention include a neurotropic adeno-associated viral vector such as a neurotropic recombinant adeno-associated virus (rAAV).

The nucleic acids that can inhibit the expression of ERK2 can be combined with other types of ERK2 inhibitors and/or other types of pain medications.

In some embodiments, the inhibitor can be a compound of formula I:

-   -   or a pharmaceutically acceptable salt thereof, wherein:         -   A¹ is N or C¹⁰;         -   A² is N or CR¹¹;         -   T is selected from —C(R⁷)₂—, C(O)—, —C(O)C(O)—, —C(O)NR⁷—,             —C(O)NR⁷NR⁷—, —CO₂—, —OC(O)—, —NR⁷CO₂—, —O—, —NR⁷C(O)NR⁷—,             OC(O)NR⁷—, —NR⁷NR⁷—, —NR⁷C(O)—, —S—, —SO—, —SO₂—NR⁷—,             —SO₂NR⁷—, —NR⁷SO₂—, —NR⁷O₂—, or —NR⁷SO₂NR⁷—;         -   m is selected from zero or one;         -   R¹ is selected from: (a) hydrogen, CN, halogen, R, N(R⁷)₂,             OR, or OH, wherein m is zero; or (b) hydrogen or R, wherein             m is one;         -   X is selected from —C(O)—, —C(O)NR⁷—, —NR⁷C(O)—, —NR⁷SO₂—,             —SO₂NR⁷—, —S(O)—, or —SO₂—;         -   R² is selected from —(CH₂)_(y)R⁵, —(CH₂)_(y)CH(R⁵)₂,             —(CH₂)_(y)CH(R⁸)(R⁵), —(CH₂)_(y)CH(R⁸)CH(R⁵)₂, —N(R⁴)₂,             —NR⁴(CH₂)_(y)N(R⁴)₂, —ON(R⁷)₂, or —NR⁷OR⁶;         -   y is 0-6;         -   R³ is selected from —R, —OR⁶, —SR⁶, —S(O)R⁶, —SO₂R⁶,             —ON(R⁷)₂, —N(R)₂, —NRN(R⁷)₂, or —NROR⁶;         -   R⁶ is selected from hydrogen or —R;         -   each R is independently selected from an optionally             substituted group selected from C₁₋₆ aliphatic; 3-7 membered             saturated, partially saturated, or aromatic monocyclic ring             having zero to three heteroatoms independently selected from             nitrogen, sulfur, or oxygen; or an 8-10 membered saturated,             partially saturated, or aromatic bicyclic ring having zero             to four heteroatoms independently selected from nitrogen,             sulfur, or oxygen;         -   each R⁴ is independently selected from —R, —R⁷, —COR⁷,             —CO₂R, —CON(R⁷)₂,     -   —SO₂R⁷, —(CH₂)_(y)R⁵, or —(CH₂)_(y)CH(R⁵)₂;         -   each R⁵ is independently selected from —R, —OR, —CO₂R,             —(CH₂)_(y)N(R⁷)₂, —N(R⁷)₂, —OR⁷, —SR⁷, —NR⁷C(O)R⁷,             —NR⁷CON(R⁷)₂, —C(O)N(R⁷)₂, —SO₂R⁷, —NR⁷SO₂R⁷, —C(O)R⁷, —CN,             or —SO₂N(R⁷)₂;         -   each R⁷ is independently selected from hydrogen or an             optionally substituted     -   C₁₋₆ aliphatic group, or two R⁷ groups bound to the same         nitrogen are taken together with the nitrogen to form a 3-7         membered heterocyclic ring having 0-2 heteroatoms in addition to         the nitrogen, independently selected from nitrogen, oxygen, or         sulfur;         -   R⁸ is selected from —R, —(CH₂)_(w)OR⁷, —(CH₂)_(w)N(R⁴)₂, or             —(CH₂)_(w)SR⁷;         -   each w is independently selected from 0-4;         -   R⁹ is selected from hydrogen, a C₁₋₆ aliphatic group,             C(O)R⁷, C(O)OR⁷, or SO₂R⁷;         -   R¹⁰ is selected from R⁷, halogen, CN, NO₂, OR⁷, SR⁷, N(R⁷)₂,             C(O)R⁷, or CO₂R⁷; or R¹⁰ and R³ are taken together to form             an optionally substituted 5-7 membered saturated, partially             saturated, or aromatic ring having 0-2 heteroatoms             independently selected from nitrogen, oxygen, and sulfur;         -   R¹¹ is selected from R⁷, halogen, CN, NO₂, OR⁷, SR⁷, N(R⁷)₂,             C(O)R⁷, or CO₂R⁷;         -   R¹² is selected from R⁷, CN, NO₂, halogen, N(R⁷)₂, SR⁷, and             OR⁷; and         -   R¹³ is selected from R⁷, CN, NO₂, halogen, N(R⁷)₂, SR⁷, and             OR⁷;         -   provided that only one of R¹² and R¹³ is a 3-7 membered             saturated, partially saturated, or aromatic monocyclic ring             having zero to three heteroatoms independently selected from             nitrogen, sulfur, or oxygen; or an 8-10 membered saturated,             partially saturated, or aromatic bicyclic ring having zero             to four heteroatoms independently selected from nitrogen,             sulfur, or oxygen.

In other embodiments, the inhibitor can be one of the following compounds

or a combination thereof.

The compounds that inhibit ERK2 can be combined with other ERK2 inhibitors and/or other pain medications.

Another aspect of the invention is an expression cassette comprising a promoter and a polynucleotide segment comprising a DNA or RNA corresponding to any of SEQ ID NO:3-162, 166-764, 773-775, 779, 782, 785, or a combination thereof. The expression cassette can also comprise a promoter and a polynucleotide segment that can hybridize to a DNA or RNA corresponding to any of SEQ ID NO:3-162, 166-764, 773-775, 779, 782, 785, or a combination thereof. The segment in the expression cassette can have the sequence X-L-Y, wherein X is a sense sequence, L is a spacer linked to the 3′ end of the sense sequence, and Y is an antisense sequence linked to the 3′ end of the linker, and wherein the Y antisense sequence is complementary to the X sequence so that upon expression of the polynucleotide segment, a short hairpin RNA (shRNA) is generated. The sense sequence can be any of the DNA or RNA sense sequences corresponding to SEQ ID NO:3-162, 166-764, 773-775, 779, 782, 785 or a combination thereof. Such an expression cassette can be present in an expression vector. In some embodiments, the expression vector is a viral vector. For example, the viral vector can be a neurotropic adeno-associated viral vector, such as a neurotropic recombinant adeno-associated virus (rAAV).

Another aspect of the invention is a composition comprising a carrier and any of the expression cassettes described herein or any of the expression vectors described herein.

Another aspect of the invention is the use of an ERK2 inhibitor in the preparation of a medicament for the treatment, inhibition and/or prevention of pain.

DESCRIPTION OF THE FIGURES

FIGS. 1A-J illustrate expression of green fluorescent protein (GFP) and the knockdown of ERK2 mRNA in the mouse spinal cord dorsal horn (SCDH) at 3 weeks after the intraparenchymal injection of a neurotropic recombinant adeno-associated virus (rAAV) vector expressing ERK2 siRNA. The expression of GFP was observed in the ipsilateral SCDH following administration of a rAAV vector that expressed a control scrambled ERK2-7 control siRNA (MM, FIG. 1A) or an active ERK2 siRNA, 2-5 (FIG. 1B, sense strand SEQ ID NO:765), 2-7 (FIG. 1C, sense strand SEQ ID NO:769) or 2-8 (FIG. 1D, sense strand, SEQ ID NO:770). Thus, the expression of GFP was not influenced by co-expression of the ERK2 siRNAs. In situ hybridization revealed no change in ERK2 mRNA level in the ipsilateral SCDH compared to the contralateral side in animals treated with vector MM (FIG. 1E). In contrast, each of the three active siRNA vectors induces a significant decrease in ERK2 mRNA in the ipsilateral SCDH (FIG. 1F-1H). Scale bar, 500 μm. In FIG. 1I, ERK2 mRNA expression was quantified from in situ images and plotted in the bar graph as the ratio of the integrated optical density of in situ labeling in the ipsilateral SCDH to that in the contralateral SCDH. Data are the mean±SEM (n=3). Compared to the control vector MM, siRNA vectors 2-5, 2-7 and 2-8 induced a significant decrease (*p<0.05) in the ERK2 mRNA expression in the ipsilateral SCDH. There was no significant difference in the ratio among the three active siRNA vectors. FIG. 1J shows that a psiCHECK luciferase assay confirms that shRNA sequences expressed by a rAAV vector are processed to siRNAs that are active against a fusion mRNA of the ERK2 and the humanized Renilla luciferase (hRluc) gene. HEK 293 cells were cotransfected with the psiCHECK plasmid (which contains the ERK2-Renilla luciferase fusion gene and the firefly luciferase gene) and with a rAAV plasmid expressing a candidate ERK2 shRNA sequence. rAAV plasmids that yield active siRNAs reduce the expression of Renilla luciferase relative to firefly luciferase, leading to a reduction of the activity ratio. The assay results are the mean±SEM (n=4). The Renilla/firefly signal was normalized to a value of 100% from the results of a concurrent assay containing psiCHECK and a rAAV plasmid that lacked coding for a shRNA sequence (GFP). Significant reductions (*p<0.05) in the ratio were observed for each of the three rAAVs plasmids encoding siRNAs (2-5, 2-7 and 2-8). A rAAV plasmid that contained a mismatch (MM) sequence of the active siRNA 2-7 was ineffective in this assay.

FIGS. 2A to 2G1-G9 illustrate inhibition of ERK2 expression and phosphorylation in the ipsilateral spinal cord dorsal horn (SCDH) of mice treated with vector 2-7, that expresses an active ERK2 siRNA with sense strand SEQ ID NO:769. An example of a blot is shown in FIG. 2A, the relative protein levels of ERK2 are shown in FIG. 2B, and the relative protein levels of ERK1 are shown in FIG. 2C. FIG. 2A shows Western blot analysis that is graphically summarized in FIG. 2B. As shown in FIG. 2A-B, reduction of ERK2 expression (*p<0.05 vs. the control vector MM group) occurs only on the ipsilateral side of the SCDH after vector 2-7 treatment. FIGS. 2A and 2C also show that ERK1 expression was unaffected by vector 2-7. Western blot analysis (FIG. 2D), graphed in FIG. 2E, showed a decrease (*p<0.05 vs. the control vector MM group) in phospho-ERK2 (pERK2) and an increase (*p<0.05 vs. the control vector MM group) in pERK1 (FIGS. 2D and F) in the ipsilateral SCDH after vector 2-7 treatment. The blot is shown in FIG. 2D, and the relative levels of pERK2 and pERK1 are shown in FIGS. 2E and 2F, respectively. FIG. 2G1-G9 show immunolabeling of sections from the spinal cord dorsal horn of mice that did not receive vector (Untreated, FIG. G1-G3)) compared with sections obtained three weeks following administration of vector MM (FIG. G4-G6) or vector 2-7 (FIG. G7-G9). Sections labeled with NeuN (FIGS. G1, G4, G7), GFAP (FIGS. G2, G5, G8) and OX42 (FIG. G3, G6, G9) show no evidence of neuronal loss or glial activation after vector administration.

FIGS. 3A to 3H1-H9 illustrate the cellular localization of pERK1/2 immunolabeling in the ipsilateral spinal cord dorsal horn (SCDH) at 3 weeks after rAAV vector administration. FIG. 3A shows that phosphorylated ERK1/2 (pERK1/2) was detected by immunolabeling in the ipsilateral SCDH after administration of the control vector, MM (red). FIG. 3B shows that NeuN labeling revealed typically distributed neuronal morphologies (green) of the same section. FIG. 3C shows the merged image, illustrating that pERK1/2 was strongly colocalized with NeuN (yellow; arrows). FIG. 3D shows that pERK1/2 immunolabeling was significantly reduced by administration of siRNA vector 2-7 (that expresses an active ERK2 siRNA with sense strand SEQ ID NO:769) compared to control vector MM (FIG. 3A). FIG. 3E shows that the NeuN labeling pattern in siRNA vector 2-7 treated SCDH is similar to that seen with vector MM (FIG. 3B). FIG. 3F shows the merged image illustrating almost no colocalization of pERK1/2 and NeuN labeling after vector 2-7 administration compared to SCDH treated with the control vector MM (FIG. 3C). Scale bar, 100 μm. FIG. 3G1-G9 illustrate the cellular localization of GFP immunolabeling in the ipsilateral SCDH 3 weeks after control MM vector administration. FIG. 3H1-3H9 illustrate the cellular localization of GFP immunolabeling in the ipsilateral SCDH three weeks after administration of vector 2-7, which expresses an active ERK2 siRNA. GFP immunolabeling was co-localized with NeuN (arrows) but not with GFAP or OX42. Scale bar, 100 μm.

FIGS. 4A-F show that siRNA vector 2-7, but not the control MM vector, reduces ERK2 expression in the ipsilateral spinal cord dorsal horn (SCDH) and prevents the phosphorylation of ERK2 that is typically induced by intraplantar administration of Complete Freund's adjuvant (CFA). FIGS. 4A and B shows Western blot analysis illustrating significant reduction in ERK2 in the ipsilateral SCDH after vector 2-7 administration compared to administration of the control MM vector in the absence of intraplantar treatment (NT) (*p<0.05, vs. vector MM/NT). This reduction in ERK2 persists from 1 to 96 hours after CFA administration (*p<0.05, vs. vector MM). Note also that at 96 hr after CFA, ERK2 is increased in the ipsilateral SCDH of the vector MM group NT compared to control (MM) ERK2 levels 1 hr after CFA (#p<0.05). FIGS. 4A and C show that ERK1 expression is also increased at 96 hr after CFA administration (#p<0.05) and this increase is prevented by vector 2-7 treatment. FIGS. 4D and 4E show that pERK2 is reduced after siRNA vector 2-7 administration and that this reduction persists from 1 hour to 96 hours after CFA (*p<0.05, vs. vector MM). In animals receiving the control vector (MM), pERK2 was increased at 1 and 96 hr after CFA administration, compared to the no treatment control (#p<0.05). FIG. 4C shows that ERK1 was increased at 96 hours after administration of CFA (*p<0.05, vs. vector MM/NT). This increase in ERK1 was not observed after CFA in animals administered the ERK2 siRNA vector 2-7. FIG. 4F shows that pERK1 was increased at 1 and 96 hr in the control (vector MM group; #p<0.05 vs. vector MM/NT), but that pERK1 levels remained relatively unchanged after administration of CFA and the ERK2 siRNA vector 2-7.

FIG. 5A-F show that the control vector MM did not change the expression or phosphorylation of ERK1 and ERK2 compared to mice that did not receive treatment (NT). FIG. 5A is an example of the Western blot of ERK1 and 2 as well as pERK1 and 2 proteins. FIG. 5B-5E are bar graphs quantifying the amounts of these proteins and revealing that no change in ERK2 expression (FIG. 5B), ERK1 expression (FIG. 5C), pERK2 (FIG. 5D) or pERK1 (FIG. 5E) occurs upon administration of the control MM vector, which encodes a scrambled siRNA sequence. FIG. 5F illustrates the time course of the increase in pERK2 in the ipsilateral SCDH following intraplantar CFA administration. Tissue was collected from groups (n=3) of control mice (no intraparenchymal injection (IPI) of vector) before and at 1, 24 and 96 hrs after intraplantar CFA. The proteins from these tissues were subjected to Western blot analysis and probed for pERK and actin. FIG. 5F shows the ratios of pERK2/actin normalized to the before CFA administration control. The expression of pERK 2 protein was increased at 1 hr and that increase is sustained through 24 and 96 hrs post CFA. (*p<0.05 vs. the before treatment group).

FIGS. 6A-H show that ERK2 siRNA vector 2-7 prevents the increased phosphorylation of ERK1/2 that is induced in the ipsilateral spinal cord dorsal horn (SCDH) by intraplantar administration of Complete Freund's adjuvant (CFA). FIG. 6A shows the basal level expression of pERK1/2 can be seen mainly in lamina I-II neurons in the control mice after administration of control vector MM and in the absence of intraplantar CFA treatment (NT). FIGS. 6B-6C illustrates that pERK1/2 immunolabeling was increased at 1 hour (FIG. 6B) and 96 hours (FIG. 6C) following CFA administration. FIGS. 6D-6F show that pERK1/2 immunolabeling in the SCDH of mice treated with vector 2-7 is reduced compared to the MM control vector, before treatment (NT) and after CFA administration. FIGS. 6G and H show that quantification of pERK1/2 as labeled neuron density (FIG. 6G) or percentage of field (FIG. 6H) in laminas I-II revealed a significant increase in pERK1/2 at 1 hour and 96 hours after CFA administration the MM control mice (#p<0.05 vs. vector MM/NT). Vector 2-7 reduced pERK1/2 immunolabeling at each corresponding time point compared to vector MM (FIGS. 6G and 6H, *p<0.05, vs. vector MM). CFA induced an increase in pERK1/2 labeling as measured by neuron density or percentage of field at 1 hour and 96 hours in the control vector MM group (FIGS. 6G and 6H, #p<0.05). For the vector 2-7 group, CFA produced increases in labeling although less than the corresponding increases in the vector MM group. Scale bar, 500 μm.

FIGS. 7A-B graphically illustrate that a spatial knockdown of ERK2 in the spinal cord dorsal horn (SCDH) by siRNA vector 2-7 does not affect thermal (FIG. 7A) or mechanical (FIG. 7B) paw-withdrawal thresholds. A brief thermal stimulus was applied to the paw (FIG. 7A), or mechanical (tactile) stimuli were applied using von Frey hairs (FIG. 7B). These thresholds were measured before and 3 weeks after the ipsilateral intraparenchymal injection of vector into the right SCDH of either the control vector (MM) or the siRNA vector 2-7 (n=10 per treatment group).

FIGS. 8A-C illustrate that a spatial knockdown of ERK2 in the spinal cord dorsal horn (SCDH) by vector 2-7 significantly reduces thermal hyperalgesia (FIG. 8B) and mechanical allodynia (FIG. 8C) resulting from the intraplantar injection of the inflammatory agent, Complete Freund's adjuvant (CFA). FIG. 8A shows that CFA administration resulted in equivalent inflammation as measured by paw size at 24, 48 and 96 hr (*p<0.05, vs. baseline, n=10 per treatment group). FIG. 8B illustrates thermal withdrawal latency (thermal hyperalgesia) of mice who had received either vector MM or vector 2-7. FIG. 8C illustrates mechanical withdrawal threshold (mechanical allodynia) who had received either vector MM or vector 2-7. The control vector MM group showed thermal hyperalgesia as measured by a reduction in the paw withdrawal threshold using a thermal stimulus (FIG. 8B) and mechanical allodynia as measured by a reduction in the mechanical threshold (50% g threshold) using von Frey hairs (FIG. 8C) applied to the paw at 24, 48 and 96 hr after intraplantar CFA compared to the baseline (* p<0.05, vs. baseline). The mice that received vector 2-7 were protected from CFA-induced thermal hyperalgesia (FIG. 8B) and mechanical allodynia (FIG. 8C) (n=10 per treatment group). Error bars indicate SEM.

FIG. 9A-9G illustrate immunolabeling of c-fos in the ipsilateral SCDH following intraplantar CFA administration. FIG. 9A shows that in the vector control animals, c-fos immunolabeling is absent from the SCDH when no CFA is administered. FIGS. 9B and 9C show that CFA induced c-fos expression in the ipsilateral SCDH at 1 hour (FIG. 9B) but not at 96 hour (FIG. 9C) after CFA administration. FIG. 9E shows that CFA induced less c-fos expression at 1 hour in animals that received the ERK2 siRNA vector 2-7. FIG. 9F shows the c-fos expression observed at 96 hours after CFA injection in animals that received the ERK2 siRNA vector 2-7. FIG. 9D shows the basal level of c-fos expression in animals that received the ERK2 siRNA vector 2-7 when no CFA is administered. FIG. 9G graphically illustrates quantification of c-fos labeled neurons in lamina I-II, revealing a significant increase at 1 hour after CFA administration (*p<0.05 vs. no treatment (NT)). However, administration of the ERK2 siRNA vector 2-7 reduced the degree to which c-fos expression increased.

FIG. 10A-H illustrate that dynorphin A immunolabeling in the ipsilateral SCDH was not changed by intraplantar CFA administration after 1 hour or even 96 hours. FIGS. 10A-C show Dynorphin A immunolabeling in the absence of CFA (FIG. 10A), at 1 hr (FIG. 10B) or 96 hr (FIG. 10C) after CFA administration in the MM vector control animals. FIGS. 10D-F show Dynorphin A immunolabeling in the absence of CFA (FIG. 10D), at 1 hr (FIG. 10E) or 96 hr (FIG. 10F) after CFA administration of the ERK2 siRNA vector 2-7 to animals. Quantification of dynorphin A labeled neurons (FIG. 10G) or percentage of field labeled with dynorphin A (FIG. 10H) revealed no significant changes following CFA in either group of animals at 1 hr or 96 hr.

FIG. 11A-B illustrate that siRNAs directed against human ERK2 dramatically reduce expression of human ERK2 in cultured human embryonic kidney (HEK293) cells. FIG. 11A is an image of a Western blot showing that each of the human ERK2 siRNAs #1 (SEQ ID NO:773), #2 (SEQ ID NO:774) and #3 (SEQ ID NO:775) significantly reduced human ERK2 expression without significantly changing ERK1 expression. FIG. 11B graphically illustrates the reduction of human ERK2 expression by each of the human ERK2 siRNAs. As shown, each of human ERK2 siRNAs #1 (SEQ ID NO:773), #2 (SEQ ID NO:774) and #3 (SEQ ID NO:775) reduced human ERK2 expression by about 70-80%.

FIG. 12A-B illustrates the construction of nucleic acids that encode an shRNA. FIG. 12A is a schematic diagram of the structure of an shRNA cassette, with the non-template strand shown. The sequence is shown in the 5′ to 3′ direction, where xx represent additional residues (1-5 residues) that are used for cloning (i.e., adaptor sequences to join the cassette to the vector, which are typically partial sequence of a restriction endonuclease site). FIG. 12B shows a structure of a vector that can express the shRNA cassette shown in FIG. 12A.

DETAILED DESCRIPTION OF THE INVENTION

This application describes agents, compositions and methods for reducing and/or inhibiting pain that involve use of ERK2 inhibitors.

Pain

The compositions and methods herein are useful for treating and/or reducing pain. All types of pain can be treated with the compositions and methods, including chronic pain, acute pain (e.g., nociceptive pain), inflammatory pain, somatic pain, visceral pain, neuropathic pain, and combinations thereof.

There are primarily three types of pain: somatic, visceral and neuropathic, all of which can be acute and chronic.

Somatic pain is typically caused by the activation of pain receptors in either the cutaneous or musculoskeletal tissues. In contrast to surface somatic pain which is usually described as sharp and may have a burning or pricking quality, deep somatic pain is usually characterized as a dull, aching but localized sensation. Somatic pain may include fractures in the vertebrae, joint pain (deep somatic pain) and postsurgical pain from a surgical incision (surface pain). Thus, the pain to be treated can be a form of somatic pain.

Visceral pain is caused by activation of pain receptors in internal areas of the body that are enclosed within a cavity. Visceral pain is usually described as pressure-like, poorly localized and deep. Therefore, the pain to be treated can be a form of visceral pain.

Neuropathic pain, caused by neural damage, is usually described as burning, tingling, shooting or stinging but can also manifest itself as sensory loss either as a result of compression, infiltration, chemical or metabolic damage or is idiopathic. Examples of neuropathic pain are heterogenous and include medication-induced neuropathy and nerve compression syndromes such as carpal tunnel, radiculopathy due to vertebral disk herniation, post-amputation syndromes such as stump pain and phantom limb pain, metabolic disease such as diabetic neuropathy, neurotropic viral disease from herpes zoster and human immunodeficiency virus (HIV) disease, tumor infiltration leading to irritation or compression of nervous tissue, radiation neuritis, as after cancer radiotherapy, and autonomic dysfunction from complex regional pain syndrome (CRPS). Thus, the pain to be treated can be a form of neuropathic pain.

Inflammatory pain is related to tissue damage which can occur in the form of penetration wounds, burns, extreme cold, fractures, inflammatory arthropathies as seen in many autoimmune conditions, excessive stretching, infections, vasoconstriction and cancer. The pain to be treated can therefore be a form of inflammatory pain.

The chronic pain can be due to problems such as arthritis, cancer, injuries, HIV, and the like. According to the invention, the compositions and methods can treat chronic pain.

Acute pain, termed nociception, is the instantaneous onset of a painful sensation in response to a noxious stimulus. It is considered to be adaptive because it can prevent an organism from damaging itself. For example, removing a hand from a hot stove as soon as pain is felt can prevent serious burns. The second type of pain is persistent pain. Unlike acute pain, it usually has a delayed onset but can last for hours to days. It is predominately considered adaptive because the occurrence of persistent pain following injury can prevent further damage to the tissue. For example, the pain associated with a sprained ankle will prevent the patient from using the foot, thereby preventing further trauma and aiding healing. A third category of pain is chronic pain. It has a delayed onset and can last for months to years. In contrast to acute and persistent pain, chronic pain is considered maladaptive and is associated with conditions such as arthritis, nerve injury, AIDS and diabetes. Yet another type of pain can be termed breakthrough pain. This is a brief flare-up of severe pain lasting from minutes to hours that can occur in the presence or absence of a preceding or precipitating factor even while the patient is regularly taking pain medication. Many patients experience a number of episodes of breakthrough pain each day. The pain to be treated with the compositions and methods described herein can be acute pain.

According to the invention, pain can be treated or inhibited in an animal. As used herein an animal is a mammal or a bird. Thus, animals that can be treated using the compositions and/or methods of the invention include humans, domesticated animals, experimental animals and zoo animals. For example, animals that can be treated using the compositions and/or methods of the invention include humans, dogs, cats, horses, pigs, cattle, goats, mice, rats, rabbits, and the like.

ERK2

Extracellular signal-regulated kinases ERK1 and ERK2 (Boulton et al., 1991) are also referred to as p44 and p42 mitogen-activated protein kinase (MAPK). ERK1 and ERK2 belong to a group of evolutionarily conserved serine/threonine protein kinases that play critical roles in cell proliferation, differentiation and survival. They are activated by dual phosphorylation on their regulatory tyrosine and threonine residues by an upstream kinase, MEK. In the central nervous system, ERK1 and ERK2 have been linked to signal transduction cascades that regulate neuronal activity and plasticity.

The high structural resemblance between ERK1 and ERK2 has limited studies of their individual contributions to physiological processes. ERK1 and ERK2 also have similar sensitivities to activation by MEK (Zheng and Guan, 1993) and are often functionally redundant in vitro (Robbins et al., 1993). However, studies in knockout mice indicate that ERK1 and ERK2 can play different roles in vivo. For example, ERK1 knockout mice are viable, fertile and of normal size (Pages et al., 1999), but ERK2 knockout mice die before embryonic day 8.5 due to defects in trophoblast and placental development and in mesoderm differentiation (Hatano et al., 2003; Saba-El-Leil et al., 2003; Yao et al., 2003). These studies suggest that ERK1 is dispensable as long as ERK2 can compensate for its loss. However, the converse is not true—ERK2 is an essential protein. Other studies indicate that ERK1 and ERK2 respond differently to growth factors and may regulate cell proliferation differently (Li and Johnson, 2006)(Fremin et al., 2007)(Zeng et al., 2005). Furthermore, ERK2 (but not ERK1) is involved in the modulation of hippocampal long term potentiation (English and Sweatt, 1996).

As shown in the Examples of this application, small interfering RNAs (siRNA) that hybridize to ERK2 mRNA selectively knock down the expression of ERK2 in spinal cord dorsal horn neurons. The siRNA was delivered by a neurotropic recombinant adeno-associated virus (rAAV), which limited the knockdown of ERK2 to neurons, and permitted examination of the specific role of the neuronal spinal cord dorsal horn ERK2 in the development of injury-induced pain hypersensitivity in vivo. Such reduction of ERK2 expression protected animals from developing mechanical allodynia (a painful response to what usually would be a non-painful stimulus) and thermal hyperalgesia (increased sensation to painful stimuli that accompany thermal injury) throughout the 96 hr after CFA.

Sequences for ERK2 proteins are readily available, for example, from the website provided by the National Center for Biotechnology Information (NCBI) at www.ncbi.nlm.nih.gov. One example of a human ERK2 sequence is provided below as SEQ ID NO:1 (NCBI accession number NP_002736 (gi:66932916)).

   1 MAAAAAAGAG PEMVRGQVFD VGPRYTNLSY IGEGAYGMVC   41 SAYDNVNKVR VAIKKISPFE HQTYCQRTLR EIKILLRFRH   81 ENIIGINDII RAPTIEQMKD VYIVQDLMET DLYKLLKTQH  121 LSNDHICYFL YQILRGLKYI HSANVLHRDL KPSNLLLNTT  161 CDLKICDFGL ARVADPDHDH TGFLTEYVAT RWYRAPEIML  201 NSKGYTKSID IWSVGCILAE MLSNRPIFPG KHYLDQLNHI  241 LGILGSPSQE DLNCIINLKA RNYLLSLPHK NKVPWNRLFP  281 NADSKALDLL DKMLTFNPHK RIEVEQALAH PYLEQYYDPS  321 DEPIAEAPFK FDMELDDLPK EKLKELIFEE TARFQPGYRS One example of a nucleotide sequence for the SEQ ID NO:1 ERK2 protein is provided below as SEQ ID NO:2 (NCBI accession number NM_138957, gi:75709179).

   1 GCCCCTCCCT CCGCCCGCCC GCCGGCCCGC CCGTCAGTCT   41 GGCAGGCAGG CAGGCAATCG GTCCGAGTGG CTGTCGGCTC   81 TTCAGCTCTC CCGCTCGGCG TCTTCCTTCC TCCTCCCGGT  121 CAGCGTCGGC GGCTGCACCG GCGGCGGCGC AGTCCCTGCG  161 GGAGGGGCGA CAAGAGCTGA GCGGCGGCCG CCGAGCGTCG  201 AGCTCAGCGC GGCGGAGGCG GCGGCGGCCC GGCAGCCAAC  241 ATGGCGGCGG CGGCGGCGGC GGGCGCGGGC CCGGAGATGG  281 TCCGCGGGCA GGTGTTCGAC GTGGGGCCGC GCTACACCAA  321 CCTCTCGTAC ATCGGCGAGG GCGCCTACGG CATGGTGTGC  361 TCTGCTTATG ATAATGTCAA CAAAGTTCGA GTAGCTATCA  401 AGAAAATCAG CCCCTTTGAG CACCAGACCT ACTGCCAGAG  441 AACCCTGAGG GAGATAAAAA TCTTACTGCG CTTCAGACAT  481 GAGAACATCA TTGGAATCAA TGACATTATT CGAGCACCAA  521 CCATCGAGCA AATGAAAGAT GTATATATAG TACAGGACCT  561 CATGGAAACA GATCTTTACA AGCTCTTGAA GACACAACAC  601 CTCAGCAATG ACCATATCTG CTATTTTCTC TACCAGATCC  641 TCAGAGGGTT AAAATATATC CATTCAGCTA ACGTTCTGCA  681 CCGTGACCTC AAGCCTTCCA ACCTGCTGCT CAACACCACC  721 TGTGATCTCA AGATCTGTGA CTTTGGCCTG GCCCGTGTTG  761 CAGATCCAGA CCATGATCAC ACAGGGTTCC TGACAGAATA  801 TGTGGCCACA CGTTGGTACA GGGCTCCAGA AATTATGTTG  841 AATTCCAAGG GCTACACCAA GTCCATTGAT ATTTGGTCTG  881 TAGGCTGCAT TCTGGCAGAA ATGCTTTCTA ACAGGCCCAT  921 CTTTCCAGGG AAGCATTATC TTGACCAGCT GAACCACATT  961 TTGGGTATTC TTGGATCCCC ATCACAAGAA GACCTGAATT 1001 GTATAATAAA TTTAAAAGCT AGGAACTATT TGCTTTCTCT 1041 TCCACACAAA AATAAGGTGC CATGGAACAG GCTGTTCCCA 1081 AATGCTGACT CCAAAGCTCT GGACTTATTG GACAAAATGT 1121 TGACATTCAA CCCACACAAG AGGATTGAAG TAGAACAGGC 1161 TCTGGCCCAC CCATATCTGG AGCAGTATTA CGACCCGAGT 1201 GACGAGCCCA TCGCCGAAGC ACCATTCAAG TTCGACATGG 1241 AATTGGATGA CTTGCCTAAG GAAAAGCTCA AAGAACTAAT 1281 TTTTGAAGAG ACTGCTAGAT TCCAGCCAGG ATACAGATCT 1321 TAAATTTGTC AGGTACCTGG AGTTTAATAC AGTGAGCTCT 1361 AGCAAGGGAG GCGCTGCCTT TTGTTTCTAG AATATTATGT 1401 TCCTCAAGGT CCATTATTTT GTATTCTTTT CCAAGCTCCT 1441 TATTGGAAGG TATTTTTTTA AATTTAGAAT TAAAAATTAT 1481 TTAGAAAGTT ACATATAAA Inhibitors of ERK2

According to the invention, any ERK2 inhibitor can be used to treat or reduce pain in an animal (e.g., in a human). Such ERK2 inhibitors can be nucleic acids that inhibit the expression of ERK2 protein, small molecule ERK2 inhibitors, anti-ERK2 antibodies and combinations thereof. These types of ERK2 inhibitors are described in more detail below.

The ERK2 inhibitor(s) employed in the compositions and methods described herein can partially or completely inhibit ERK2. Thus, the ERK2 inhibitor(s) can inhibit about 99% ERK2 activity or expression, or about 95% ERK2 activity or expression, or about 90% ERK2 activity or expression, or about 80% ERK2 activity or expression, or about ERK2 60% activity or expression, or about 50% ERK2 activity or expression, or about 35% ERK2 activity or expression, or any level if inhibition greater than about 30% ERK2 inhibition. Moreover, administered locally, the percent ERK2 inhibition may in some embodiments be greater than would be desirable when ERK2 is administered systemically. Thus, for example, when administered locally (e.g., to the site of pain or to the spinal fluid or column), the ERK2 inhibition can be more than 95% ERK2 inhibition, or more than 90% ERK2 inhibition, or more than 85% ERK2 inhibition, or more than 80% ERK2 inhibition, or more than 75% ERK2 inhibition, or more than 70% ERK2 inhibition, or more than 65% ERK2 inhibition, or more than 60% ERK2 inhibition, or more than 50% ERK2 inhibition. However, in other embodiments when ERK2 inhibitors are administered systemically, a lesser percent ERK2 inhibition may be desirable. For example, when administered systemically, the ERK2 inhibition can be less than 60% ERK2 inhibition, or less than 55% ERK2 inhibition, or less than 50% ERK2 inhibition, or less than 40% ERK2 inhibition, or less than 30% ERK2 inhibition.

While the focus of the methods is upon inhibiting ERK2, some inhibition of ERK1 may occur when using some of these ERK2 inhibitors because ERK1 and ERK2 are so closely related. Some inhibition of ERK1 is acceptable. For example, while ERK2 knockout mice die before embryonic day 8.5, mutations that lead to complete loss of ERK1 function do not affect the viability, fertility and or growth of animals. Thus, in some embodiments, ERK1 is inhibited to about the same extent as, or to an even a greater extent than, ERK2 by a selected ERK2 inhibitor or combination of inhibitors. In other embodiments, ERK1 is inhibited less than ERK2 by a selected ERK2 inhibitor or combination of inhibitors. For example, administration of an ERK2 inhibitor can in some embodiments give rise to less than 70% ERK1 inhibition, or less than 60% ERK1 inhibition, or less than 50% ERK1 inhibition, or less than 40% ERK1 inhibition, or less than 30% ERK1 inhibition, or less than 20% ERK1 inhibition, or less than 10% ERK1 inhibition.

Nucleic Acid Inhibitors

In some embodiments, the ERK2 inhibitors used in the compositions and methods described herein are nucleic acids that can inhibit the expression of an ERK2 protein. Nucleic acids that can inhibit the expression of an ERK2 protein include small interfering RNAs (siRNAs), ribozymes, antisense nucleic acids, and the like. For example, small interfering RNAs (siRNA) targeted against ERK2 transcripts were used to specifically reduce ERK2 expression by about 75% to 80% (see Example 2).

In some embodiments, an inhibitory nucleic acid of the invention can hybridize to an ERK2 nucleic acid (e.g., any of SEQ ID NOs: 2 or SEQ ID NO:771) under intracellular conditions. In other embodiments, the inhibitory nucleic acids can hybridize to an ERK2 nucleic acid under stringent hybridization conditions. In general, the term “hybridize” is used to indicate that a nucleic acid specifically hybridizes to a complementary nucleic acid.

The inhibitory nucleic acids of the invention are sufficiently complementary to endogenous ERK2 nucleic acids to inhibit expression of an ERK2 nucleic acid under either intracellular conditions or under string hybridization conditions. In many embodiments it is desirable for ERK2 inhibitory nucleic acids to hybridize to ERK2 mRNA (e.g., the mRNA encoded by SEQ ID NO:2 or SEQ ID NO:771). However, the ERK2 inhibitory nucleic acid need not be 100% complementary to an endogenous ERK2 mRNA. Instead the ERK2 inhibitory nucleic acid can be less than 100% complementary to an endogenous ERK2 mRNA. For example, the ERK2 inhibitory nucleic acid can have one, two, three, four, or five mismatches or nucleotides that are not complementary to an endogenous ERK2 mRNA.

Intracellular conditions refer to conditions such as temperature, pH and salt concentrations typically found inside a cell, e.g. a mammalian cell. One example of such a mammalian cell is a neuron.

Generally, stringent hybridization conditions are selected to be about 5° C. lower than the thermal melting point (T_(m)) for the specific sequence at a defined ionic strength and pH. However, stringent conditions encompass temperatures in the range of about 1° C. to about 20° C. lower than the thermal melting point of the selected sequence, depending upon the desired degree of stringency as otherwise qualified herein.

In some embodiments, an ERK2 inhibitory nucleic acid has a stretch of 10, 11, 12, 13, 14, 15, 16, 17, or 18 contiguous nucleotides that are complementary to an ERK2 DNA or RNA. However, inhibitory nucleic acids that comprise, for example, 2, 3, 4, or 5 or more stretches of contiguous nucleotides that are precisely complementary to an ERK2 coding sequence, each separated by a stretch of contiguous nucleotides that are not complementary to adjacent coding sequences, may also inhibit the function of a ERK2 nucleic acid. In general, each stretch of contiguous, complementary nucleotides is at least 4, 5, 6, 7, or 8 or more nucleotides in length. Non-complementary intervening sequences may be 1, 2, 3, or 4 nucleotides in length. One skilled in the art can easily use the calculated melting point of an inhibitory nucleic acid hybridized to an ERK2 nucleic acid to estimate the degree of mismatching that will be tolerated for inhibiting expression of ERK2. Inhibitory nucleic acids of the invention include, for example, a small interfering RNA, a ribozyme or an antisense nucleic acid molecule.

An antisense nucleic acid molecule may be single or double stranded (e.g. a small interfering RNA (siRNA)), and may function in an enzyme-dependent manner or by steric blocking.

Small interfering RNA (siRNA) molecules are also called short interfering RNA or silencing RNA. These siRNA molecules are double-stranded and are generally about 20-25 nucleotides in length, with a two to three nucleotide overhang on one or both ends. Typically, siRNA interfere with gene expression by binding to mRNA, which leads to degradation of the mRNA by nucleases. Thus, siRNAs interfere with gene expression. By selecting a sequence for the siRNA that is complementary to the mRNA transcribed by a gene of interest, the siRNA can specifically interfere with the expression from that gene. Accordingly, one aspect of the invention is an siRNA that binds to an ERK2 mRNA and interferes with (inhibits) the expression of the ERK2 protein.

siRNAs can be exogenously introduced into cells by various methods. While siRNAs can be introduced by parenteral injection, the siRNA can also be encoded within and expressed by an appropriate expression vector. This can be done by introducing a loop between the two strands of the siRNA, so that a single long transcript is expressed that naturally folds into a short hairpin RNA (shRNA). This shRNA is naturally processed into a functional siRNA within a cell. Further information on delivery of siRNAs, ribozymes, antisense RNA and the like is provided below.

The nucleotide sequence of siRNAs may be designed using a siRNA design computer program. For example, siRNA sequences may be designed using the siRNA design program (http://jura.wi.mit.edu/siRNAext/) from the Whitehead Institute for Biomedical Research (MIT)(see, Yuan et al., Nuc. Acids Res. 32:W130-134 (2004). Alternatively, siRNA sequences can be designed using a program available from the Ambion website (ambion.com/techlib/misc/siRNA finder.html).

In general, these programs generate siRNA sequences from an input DNA sequence or an input accession number (e.g., an ERK2 nucleic acid such as SEQ ID NO:2 or an NCBI accession number NM_138957) using siRNA generation rules developed as described, for example, Yuan et al. Nuc. Acids Res. 32:W130-134 (2004).

Thus, the inventors have identified the following siRNAs that bind ERK2 and can be used to inhibit, treat or reduce pain, where the targeted ERK2 mRNA sequence is identified as “mRNA,” the sense strand of the siRNA is identified as the “S” strand and where the antisense strand of the siRNA is identified as the “AS” strand. Note that thymidine (T) is used in the mRNA sequences shown below—for actual mRNA sequences, each thymidine would be a uridine (U).

S 5′:    GCAGGAGCUUGUGGAAAUA UU (SEQ ID NO: 3) mRNA: GA GCAGGAGCTTGTGGAAATA CC (SEQ ID NO: 4) AS 3′: UU CGUCCUCGAACACCUUUAU (SEQ ID NO: 5) S 5′:    GCUGCAUUCUGGCAGAAAU UU (SEQ ID NO: 6) mRNA: AG GCTGCATTCTGGCAGAAAT GC (SEQ ID NO: 7) AS 3′: UU CGACGUAAGACCGUCUUUA (SEQ ID NO: 8) S 5′:    GUGCUCUGCUUAUGAUAAU UU (SEQ ID NO: 9) mRNA: GT GTGCTCTGCTTATGATAAT GT (SEQ ID NO: 10) AS 3′: UU CACGAGACGAAUACUAUUA (SEQ ID NO: 11) S 5′:    CGUGCAUGUAUAGUUUAAU UU (SEQ ID NO: 12) mRNA: AC CGTGCATGTATAGTTTAAT TG (SEQ ID NO: 13) AS 3′: UU GCACGUACAUAUCAAAUUA (SEQ ID NO: 14) S 5′:    GUCCUCAAGUACUCAAAUA UU (SEQ ID NO: 15) mRNA: CT GTCCTCAAGTACTCAAATA TT (SEQ ID NO: 16) AS 3′: UU CAGGAGUUCAUGAGUUUAU (SEQ ID NO: 17) S 5′:    CCGUGCAUGUAUAGUUUAA UU (SEQ ID NO: 18) mRNA: CA CCGTGCATGTATAGTTTAA TT (SEQ ID NO: 19) AS 3′: UU GGCACGUACAUAUCAAAUU (SEQ ID NO: 20) S 5′:    GGUGCCUUCUUGGUAUUGU UU (SEQ ID NO: 21) mRNA: TT GGTGCCTTCTTGGTATTGT AC (SEQ ID NO: 22) AS 3′: UU CCACGGAAGAACCAUAACA (SEQ ID NO: 23) S 5′:    GAGGAACACUGCGUCUUUA UU (SEQ ID NO: 24) mRNA: GA GAGGAACACTGCGTCTTTA AA (SEQ ID NO: 25) AS 3′: UU CUCCUUGUGACGCAGAAAU (SEQ ID NO: 26) S 5′:    GUGGUCACUUGUACCAUAU UU (SEQ ID NO: 27) mRNA: CT GTGGTCACTTGTACCATAT AG (SEQ ID NO: 28) AS 3′: UU CACCAGUGAACAUGGUAUA (SEQ ID NO: 29) S 5′:    CCCAAGUUUAAGGGAAAUA UU (SEQ ID NO: 30) mRNA: AT CCCAAGTTTAAGGGAAATA TT (SEQ ID NO: 31) AS 3′: UU GGGUUCAAAUUCCCUUUAU (SEQ ID NO: 32) S 5′:    CAGCCAUUCAGAGGAAACU UU (SEQ ID NO: 33) mRNA: TT CAGCCATTCAGAGGAAACT GT (SEQ ID NO: 34) AS 3′: UU GUCGGUAAGUCUCCUUUGA (SEQ ID NO: 35) S 5′:    GUGGGAUGGAAUUGAAAGA UU (SEQ ID NO: 36) mRNA: CA GTGGGATGGAATTGAAAGA AC (SEQ ID NO: 37) AS 3′: UU CACCCUACCUUAACUUUCU (SEQ ID NO: 38) S 5′:    GGCUCUUCUUACAUUUGUA UU (SEQ ID NO: 39) mRNA: TT GGCTCTTCTTACATTTGTA AA (SEQ ID NO: 40) AS 3′: UU CCGAGAAGAAUGUAAACAU (SEQ ID NO: 41) S 5′:    CCGGAUAACACUGAUUAGU UU (SEQ ID NO: 42) mRNA: TA CCGGATAACACTGATTAGT CA (SEQ ID NO: 43) AS 3′: UU GGCCUAUUGUGACUAAUCA (SEQ ID NO: 44) S 5′:    CACCAACCAUCGAGCAAAU UU (SEQ ID NO: 45) mRNA: AG CACCAACCATCGAGCAAAT GA (SEQ ID NO: 46) AS 3′: UU GUGGUUGGUAGCUCGUUUA (SEQ ID NO: 47) S 5′:    GGUAGUCACUAACAUAUAU UU (SEQ ID NO: 48) mRNA: AT GGTAGTCACTAACATATAT AA (SEQ ID NO: 49) AS 3′: UU CCAUCAGUGAUUGUAUAUA (SEQ ID NO: 50) S 5′:    GUGCCUUCUUGGUAUUGUA UU (SEQ ID NO: 51) mRNA: TG GTGCCTTCTTGGTATTGTA CC (SEQ ID NO: 52) AS 3′: UU CACGGAAGAACCAUAACAU (SEQ ID NO: 53) S 5′:    CAGGGUUCCUGACAGAAUA UU (SEQ ID NO: 54) mRNA: CA CAGGGTTCCTGACAGAATA TG (SEQ ID NO: 55) AS 3′: UU GUCCCAAGGACUGUCUUAU (SEQ ID NO: 56) S 5′:    CCACCUGUGAUCUCAAGAU UU (SEQ ID NO: 57) mRNA: CA CCACCTGTGATCTCAAGAT CT (SEQ ID NO: 58) AS 3′: UU GGUGGACACUAGAGUUCUA (SEQ ID NO: 59) S 5′:    CCCUUGAGCUACUUCAAAU UU (SEQ ID NO: 60) mRNA: CG CCCTTGAGCTACTTCAAAT GT (SEQ ID NO: 61) AS 3′: UU GGGAACUCGAUGAAGUUUA (SEQ ID NO: 62) S 5′:    GUGCAGAUGAGAAGCUAUA UU (SEQ ID NO: 63) mRNA: TG GTGCAGATGAGAAGCTATA AC (SEQ ID NO: 64) AS 3′: UU CACGUCUACUCUUCGAUAU (SEQ ID NO: 65) S 5′:    GCUCUGCUUAUGAUAAUGU UU (SEQ ID NO: 66) mRNA: GT GCTCTGCTTATGATAATGT CA (SEQ ID NO: 67) AS 3′: UU CGAGACGAAUACUAUUACA (SEQ ID NO: 68) S 5′:    GUCAGAAACAAAUGGAAAU UU (SEQ ID NO: 69) mRNA: GG GTCAGAAACAAATGGAAAT CC (SEQ ID NO: 70) AS 3′: UU CAGUCUUUGUUUACCUUUA (SEQ ID NO: 71) S 5′:    GCCUACGAUUGAAAUGAAA UU (SEQ ID NO: 72) mRNA: AT GCCTACGATTGAAATGAAA AC (SEQ ID NO: 73) AS 3′: UU CGGAUGCUAACUUUACUUU (SEQ ID NO: 74) S 5′:    CCCUGGUUCUCUCUAAAGA UU (SEQ ID NO: 75) mRNA: TT CCCTGGTTCTCTCTAAAGA GG (SEQ ID NO: 76) AS 3′: UU GGGACCAAGAGAGAUUUCU (SEQ ID NO: 77) S 5′:    GGGUAGAAGAAUACUGUAU UU (SEQ ID NO: 78) mRNA: CT GGGTAGAAGAATACTGTAT TG (SEQ ID NO: 79) AS 3′: UU CCCAUCUUCUUAUGACAUA (SEQ ID NO: 80) S 5′:    CCAAGUUUAAGGGAAAUAU UU (SEQ ID NO: 81) mRNA: TC CCAAGTTTAAGGGAAATAT TT (SEQ ID NO: 82) AS 3′: UU GGUUCAAAUUCCCUUUAUA (SEQ ID NO: 83) S 5′:    GGUGUGCUCUGCUUAUGAU UU (SEQ ID NO: 84) mRNA: AT GGTGTGCTCTGCTTATGAT AA (SEQ ID NO: 85) AS 3′: UU CCACACGAGACGAAUACUA (SEQ ID NO: 86) S 5′:    GAGCAAAUGAAAGAUGUAU UU (SEQ ID NO: 87) mRNA: TC GAGCAAATGAAAGATGTAT AT (SEQ ID NO: 88) AS 3′: UU CUCGUUUACUUUCUACAUA (SEQ ID NO: 89) S 5′:    CAGAGCAAGAAGUCAUAAA UU (SEQ ID NO: 90) mRNA: TT CAGAGCAAGAAGTCATAAA GA (SEQ ID NO: 91) AS 3′: UU GUCUCGUUCUUCAGUAUUU (SEQ ID NO: 92) S 5′:    GUCCUCUUCUAAAUAGAAA UU (SEQ ID NO: 93) mRNA: GA GTCCTCTTCTAAATAGAAA AC (SEQ ID NO: 94) AS 3′: UU CAGGAGAAGAUUUAUCUUU (SEQ ID NO: 95) S 5′:    CUGGCAGAAAUGCUUUCUA UU (SEQ ID NO: 96) mRNA: TT CTGGCAGAAATGCTTTCTA AC (SEQ ID NO: 97) AS 3′: UU GACCGUCUUUACGAAAGAU (SEQ ID NO: 98) S 5′:    GCCUUGUUCAAUAAUUACU UU (SEQ ID NO: 99) mRNA: TT GCCTTGTTCAATAATTACT GT (SEQ ID NO: 100) AS 3′: UU CGGAACAAGUUAUUAAUGA (SEQ ID NO: 101) S 5′:    GCUCCAGAAAUUAUGUUGA UU (SEQ ID NO: 102) mRNA: GG GCTCCAGAAATTATGTTGA AT (SEQ ID NO: 103) AS 3′: UU CGAGGUCUUUAAUACAACU (SEQ ID NO: 104) S 5′:    CCCAGCACUUGGAUUUACA UU (SEQ ID NO: 105) mRNA: GG CCCAGCACTTGGATTTACA TA (SEQ ID NO: 106) AS 3′: UU GGGUCGUGAACCUAAAUGU (SEQ ID NO: 107) S 5′:    GCCUUGUAUAUGGUAAAGA UU (SEQ ID NO: 108) mRNA: TT GCCTTGTATATGGTAAAGA TT (SEQ ID NO: 109) AS 3′: UU CGGAACAUAUACCAUUUCU (SEQ ID NO: 110) S 5′:    GCGCUAGCUAUCAUGUGUA UU (SEQ ID NO: 111) mRNA: AA GCGCTAGCTATCATGTGTA GT (SEQ ID NO: 112) AS 3′: UU CGCGAUCGAUAGUACACAU (SEQ ID NO: 113) S 5′:    GGAGUCAGAUUGGCAUGAA UU (SEQ ID NO: 114) mRNA: GT GGAGTCAGATTGGCATGAA AC (SEQ ID NO: 115) AS 3′: UU CCUCAGUCUAACCGUACUU (SEQ ID NO: 116) S 5′:    GCAUCUGGGUAGAAGAAUA UU (SEQ ID NO: 117) mRNA: TG GCATCTGGGTAGAAGAATA CT (SEQ ID NO: 118) AS 3′: UU CGUAGACCCAUCUUCUUAU (SEQ ID NO: 119) S 5′:    GGCAUUAUGUAAUGACUUA UU (SEQ ID NO: 120) mRNA: TG GGCATTATGTAATGACTTA TT (SEQ ID NO: 121) AS 3′: UU CCGUAAUACAUUACUGAAU (SEQ ID NO: 122) S 5′:    GCUCUUCUUACAUUUGUAA UU (SEQ ID NO: 123) mRNA: TG GCTCTTCTTACATTTGTAA AA (SEQ ID NO: 124) AS 3′: UU CGAGAAGAAUGUAAACAUU (SEQ ID NO: 125) S 5′:    GCUGGUGUUUGAAACAUGA UU (SEQ ID NO: 126) mRNA: AT GCTGGTGTTTGAAACATGA TA (SEQ ID NO: 127) AS 3′: UU CGACCACAAACUUUGUACU (SEQ ID NO: 128) S 5′:    GGGCACUUUAAGUCAGUGA UU (SEQ ID NO: 129) mRNA: TA GGGCACTTTAAGTCAGTGA CA (SEQ ID NO: 130) AS 3′: UU CCCGUGAAAUUCAGUCACU (SEQ ID NO: 131) S 5′:    GCAGUACUUAAUGUUUGUA UU (SEQ ID NO: 132) mRNA: GT GCAGTACTTAATGTTTGTA AG (SEQ ID NO: 133) AS 3′: UU CGUCAUGAAUUACAAACAU (SEQ ID NO: 134) S 5′:    CAGAUUAGGUCAUCUUAAU UU (SEQ ID NO: 135) mRNA: TA CAGATTAGGTCATCTTAAT TC (SEQ ID NO: 136) AS 3′: UU GUCUAAUCCAGUAGAAUUA (SEQ ID NO: 137) S 5′:    GGUGAGAAAUUUGCCUUGU UU (SEQ ID NO: 138) mRNA: GT GGTGAGAAATTTGCCTTGT TC (SEQ ID NO: 139) AS 3′: UU CCACUCUUUAAACGGAACA (SEQ ID NO: 140) S 5′:    GAAGCUAUAACAGUGAAUA UU (SEQ ID NO: 141) mRNA: GA GAAGCTATAACAGTGAATA TG (SEQ ID NO: 142) AS 3′: UU CUUCGAUAUUGUCACUUAU (SEQ ID NO: 143) S 5′:    GAAGACCUGAAUUGUAUAA UU (SEQ ID NO: 144) mRNA: AA GAAGACCTGAATTGTATAA TA (SEQ ID NO: 145) AS 3′: UU CUUCUGGACUUAACAUAUU (SEQ ID NO: 146) S 5′:    GAGCACUCAAGAAAGUUCU UU (SEQ ID NO: 147) mRNA: TA GAGCACTCAAGAAAGTTCT GA (SEQ ID NO: 148) AS 3′: UU CUCGUGAGUUCUUUCAAGA (SEQ ID NO: 149) S 5′:    CUCCAGAAAUUAUGUUGAA UU (SEQ ID NO: 150) mRNA: GG CTCCAGAAATTATGTTGAA TT (SEQ ID NO: 151) AS 3′: UU GAGGUCUUUAAUACAACUU (SEQ ID NO: 152) S 5′:    GCUUCAGACAUGAGAACAU UU (SEQ ID NO: 153) mRNA: GC GCTTCAGACATGAGAACAT CA (SEQ ID NO: 154) AS 3′: UU CGAAGUCUGUACUCUUGUA (SEQ ID NO: 155) S 5′:    GUUCCUUUAUUCACAAUCU UU (SEQ ID NO: 156) mRNA: AT GTTCCTTTATTCACAATCT TA (SEQ ID NO: 157) AS 3′: UU CAAGGAAAUAAGUGUUAGA (SEQ ID NO: 158) S 5′:    GUGUCACUCUGUAGUUACU UU (SEQ ID NO: 159) mRNA: CA GTGTCACTCTGTAGTTACT GT (SEQ ID NO: 160) AS 3′: UU CACAGUGAGACAUCAAUGA (SEQ ID NO: 161) S 5′:    GCUGUAAAGUGGAAGCAAU UU (SEQ ID NO: 162) mRNA: CA GCTGTAAAGTGGAAGCAAT AT (SEQ ID NO: 166) AS 3′: UU CGACAUUUCACCUUCGUUA (SEQ ID NO: 167) S 5′:    CCACAUGCCUACGAUUGAA UU (SEQ ID NO: 168) mRNA: TG CCACATGCCTACGATTGAA AT (SEQ ID NO: 169) AS 3′: UU GGUGUACGGAUGCUAACUU (SEQ ID NO: 170) S 5′:    GUCAGCAUCUCAAGUUCAU UU (SEQ ID NO: 171) mRNA: AT GTCAGCATCTCAAGTTCAT TT (SEQ ID NO: 172) AS 3′: UU CAGUCGUAGAGUUCAAGUA (SEQ ID NO: 173) S 5′:    GGGACACAGAAAUGUGACU UU (SEQ ID NO: 174) mRNA: AC GGGACACAGAAATGTGACT GT (SEQ ID NO: 175) AS 3′: UU CCCUGUGUCUUUACACUGA (SEQ ID NO: 176) S 5′:    CCAGUGGGAUGGAAUUGAA UU (SEQ ID NO: 177) mRNA: AG CCAGTGGGATGGAATTGAA AG (SEQ ID NO: 178) AS 3′: UU GGUCACCCUACCUUAACUU (SEQ ID NO: 179) S 5′:    CCUGCUAAUAUGAACAGAA UU (SEQ ID NO: 180) mRNA: TG CCTGCTAATATGAACAGAA AT (SEQ ID NO: 181) AS 3′: UU GGACGAUUAUACUUGUCUU (SEQ ID NO: 182) S 5′:    CUGCUAAUAUGAACAGAAA UU (SEQ ID NO: 183) mRNA: GC CTGCTAATATGAACAGAAA TG (SEQ ID NO: 184) AS 3′: UU GACGAUUAUACUUGUCUUU (SEQ ID NO: 185) S 5′:    GAGUCAGAUUGGCAUGAAA UU (SEQ ID NO: 186) mRNA: TG GAGTCAGATTGGCATGAAA CC (SEQ ID NO: 187) AS 3′: UU CUCAGUCUAACCGUACUUU (SEQ ID NO: 188) S 5′:    CUGCCUGCUAAUAUGAACA UU (SEQ ID NO: 189) mRNA: GA CTGCCTGCTAATATGAACA GA (SEQ ID NO: 190) AS 3′: UU GACGGACGAUUAUACUUGU (SEQ ID NO: 191) S 5′:    CGUCUUUAAAUGAGAAAGU UU (SEQ ID NO: 192) mRNA: TG CGTCTTTAAATGAGAAAGT AT (SEQ ID NO: 193) AS 3′: UU GCAGAAAUUUACUCUUUCA (SEQ ID NO: 194) S 5′:    GCUACACCAAGUCCAUUGA UU (SEQ ID NO: 195) mRNA: GG GCTACACCAAGTCCATTGA TA (SEQ ID NO: 196) AS 3′: UU CGAUGUGGUUCAGGUAACU (SEQ ID NO: 197) S 5′:    CAGGGAAGCAUUAUCUUGA UU (SEQ ID NO: 198) mRNA: TC CAGGGAAGCATTATCTTGA CC (SEQ ID NO: 199) AS 3′: UU GUCCCUUCGUAAUAGAACU (SEQ ID NO: 200) S 5′:    CUCCAAAGCUCUGGACUUA UU (SEQ ID NO: 201) mRNA: GA CTCCAAAGCTCTGGACTTA TT (SEQ ID NO: 202) AS 3′: UU GAGGUUUCGAGACCUGAAU (SEQ ID NO: 203) S 5′:    GCAAUGGAGAAUGGGUUAU UU (SEQ ID NO: 204) mRNA: CA GCAATGGAGAATGGGTTAT AT (SEQ ID NO: 205) AS 3′: UU CGUUACCUCUUACCCAAUA (SEQ ID NO: 206) S 5′:    GGGUCAGAAACAAAUGGAA UU (SEQ ID NO: 207) mRNA: AT GGGTCAGAAACAAATGGAA AT (SEQ ID NO: 208) AS 3′: UU CCCAGUCUUUGUUUACCUU (SEQ ID NO: 209) S 5′:    GUACCAUAUAGAGGUGUAA UU (SEQ ID NO: 210) mRNA: TT GTACCATATAGAGGTGTAA CA (SEQ ID NO: 211) AS 3′: UU CAUGGUAUAUCUCCACAUU (SEQ ID NO: 212) S 5′:    GUGGGUGUUUCAGUAACCA UU (SEQ ID NO: 213) mRNA: AT GTGGGTGTTTCAGTAACCA CG (SEQ ID NO: 214) AS 3′: UU CACCCACAAAGUCAUUGGU (SEQ ID NO: 215) S 5′:    GUAGAGCACUCAAGAAAGU UU (SEQ ID NO: 216) mRNA: GT GTAGAGCACTCAAGAAAGT TC (SEQ ID NO: 217) AS 3′: UU CAUCUCGUGAGUUCUUUCA (SEQ ID NO: 218) S 5′:    CCAGUCCUUUCAUUUAGUA UU (SEQ ID NO: 219) mRNA: TT CCAGTCCTTTCATTTAGTA TA (SEQ ID NO: 220) AS 3′: UU GGUCAGGAAAGUAAAUCAU (SEQ ID NO: 221) S 5′:    GCCUGCUAAUAUGAACAGA UU (SEQ ID NO: 222) mRNA: CT GCCTGCTAATATGAACAGA AA (SEQ ID NO: 223) AS 3′: UU CGGACGAUUAUACUUGUCU (SEQ ID NO: 224) S 5′:    CAGUGAAUAUGUGGUUUCU UU (SEQ ID NO: 225) mRNA: AA CAGTGAATATGTGGTTTCT CT (SEQ ID NO: 226) AS 3′: UU GUCACUUAUACACCAAAGA (SEQ ID NO: 227) S 5′:    GAGUCCUCUUCUAAAUAGA UU (SEQ ID NO: 228) mRNA: TG GAGTCCTCTTCTAAATAGA AA (SEQ ID NO: 229) AS 3′: UU CUCAGGAGAAGAUUUAUCU (SEQ ID NO: 230) S 5′:    GUCACUUGUACCAUAUAGA UU (SEQ ID NO: 231) mRNA: TG GTCACTTGTACCATATAGA GG (SEQ ID NO: 232) AS 3′: UU CAGUGAACAUGGUAUAUCU (SEQ ID NO: 233) S 5′:    CUAUCCAGCAGAUCAUUUA UU (SEQ ID NO: 234) mRNA: TG CTATCCAGCAGATCATTTA GG (SEQ ID NO: 235) AS 3′: UU GAUAGGUCGUCUAGUAAAU (SEQ ID NO: 236) S 5′:    CUGGGUAGAAGAAUACUGU UU (SEQ ID NO: 237) mRNA: AT CTGGGTAGAAGAATACTGT AT (SEQ ID NO: 238) AS 3′: UU GACCCAUCUUCUUAUGACA (SEQ ID NO: 239) S 5′:    CCAUCGAGCAAAUGAAAGA UU (SEQ ID NO: 240) mRNA: AA CCATCGAGCAAATGAAAGA TG (SEQ ID NO: 241) AS 3′: UU GGUAGCUCGUUUACUUUCU (SEQ ID NO: 242) S 5′:    CUGUCCUCAAGUACUCAAA UU (SEQ ID NO: 243) mRNA: TT CTGTCCTCAAGTACTCAAA TA (SEQ ID NO: 244) AS 3′: UU GACAGGAGUUCAUGAGUUU (SEQ ID NO: 245) S 5′:    GCACCAUUCAAGUUCGACA UU (SEQ ID NO: 246) mRNA: AA GCACCATTCAAGTTCGACA TG (SEQ ID NO: 247) AS 3′: UU CGUGGUAAGUUCAAGCUGU (SEQ ID NO: 248) S 5′:    CCUCAAAGCUAGCAGAGAU UU (SEQ ID NO: 249) mRNA: GT CCTCAAAGCTAGCAGAGAT AC (SEQ ID NO: 250) AS 3′: UU GGAGUUUCGAUCGUCUCUA (SEQ ID NO: 251) S 5′:    CAUGCCACGUAAUAUUUCA UU (SEQ ID NO: 252) mRNA: CA CATGCCACGTAATATTTCA GC (SEQ ID NO: 253) AS 3′: UU GUACGGUGCAUUAUAAAGU (SEQ ID NO: 254) S 5′:    CACCAUUCAAGUUCGACAU UU (SEQ ID NO: 255) mRNA: AG CACCATTCAAGTTCGACAT GG (SEQ ID NO: 256) AS 3′: UU GUGGUAAGUUCAAGCUGUA (SEQ ID NO: 257) S 5′:    GUUAUGUGCAGUACUUAAU UU (SEQ ID NO: 258) mRNA: GC GTTATGTGCAGTACTTAAT GT (SEQ ID NO: 259) AS 3′: UU CAAUACACGUCAUGAAUUA (SEQ ID NO: 260) S 5′:    CCUUGUAUAUGGUAAAGAU UU (SEQ ID NO: 261) mRNA: TG CCTTGTATATGGTAAAGAT TA (SEQ ID NO: 262) AS 3′: UU GGAACAUAUACCAUUUCUA (SEQ ID NO: 263) S 5′:    CCCAAAUGCUGACUCCAAA UU (SEQ ID NO: 264) mRNA: TT CCCAAATGCTGACTCCAAA GC (SEQ ID NO: 265) AS 3′: UU GGGUUUACGACUGAGGUUU (SEQ ID NO: 266) S 5′:    GGAUGGAAUUGAAAGAACU UU (SEQ ID NO: 267) mRNA: TG GGATGGAATTGAAAGAACT AA (SEQ ID NO: 268) AS 3′: UU CCUACCUUAACUUUCUUGA (SEQ ID NO: 269) S 5′:    CUGCGUCUUUAAAUGAGAA UU (SEQ ID NO: 270) mRNA: CA CTGCGTCTTTAAATGAGAA AG (SEQ ID NO: 271) AS 3′: UU GACGCAGAAAUUUACUCUU (SEQ ID NO: 272) S 5′:    GCCAUUCAGAGGAAACUGU UU (SEQ ID NO: 273) mRNA: CA GCCATTCAGAGGAAACTGT TT (SEQ ID NO: 274) AS 3′: UU CGGUAAGUCUCCUUUGACA (SEQ ID NO: 275) S 5′:    GUCUAGUCCUUCGUUAUGU UU (SEQ ID NO: 276) mRNA: TT GTCTAGTCCTTCGTTATGT TC (SEQ ID NO: 277) AS 3′: UU CAGAUCAGGAAGCAAUACA (SEQ ID NO: 278) S 5′:    GUGUCCCUGUAUUACCAAA UU (SEQ ID NO: 279) mRNA: TT GTGTCCCTGTATTACCAAA AT (SEQ ID NO: 280) AS 3′: UU CACAGGGACAUAAUGGUUU (SEQ ID NO: 281) S 5′:    CAGCAUGUCAGCAUCUCAA UU (SEQ ID NO: 282) mRNA: TA CAGCATGTCAGCATCTCAA GT (SEQ ID NO: 283) AS 3′: UU GUCGUACAGUCGUAGAGUU (SEQ ID NO: 284) S 5′:    GUGUAACACUUGUCAAGAA UU (SEQ ID NO: 285) mRNA: AG GTGTAACACTTGTCAAGAA GC (SEQ ID NO: 286) AS 3′: UU CACAUUGUGAACAGUUCUU (SEQ ID NO: 287) S 5′:    GUGUAGAGCACUCAAGAAA UU (SEQ ID NO: 288) mRNA: GC GTGTAGAGCACTCAAGAAA GT (SEQ ID NO: 289) AS 3′: UU CACAUCUCGUGAGUUCUUU (SEQ ID NO: 290) S 5′:    CAGCAGAUCAUUUAGGAAA UU (SEQ ID NO: 291) mRNA: TC CAGCAGATCATTTAGGAAA AA (SEQ ID NO: 292) AS 3′: UU GUCGUCUAGUAAAUCCUUU (SEQ ID NO: 293) S 5′:    CAGAUCCUCAGAGGGUUAA UU (SEQ ID NO: 294) mRNA: AC CAGATCCTCAGAGGGTTAA AA (SEQ ID NO: 295) AS 3′: UU GUCUAGGAGUCUCCCAAUU (SEQ ID NO: 296) S 5′:    GAACAUCAUUGGAAUCAAU UU (SEQ ID NO: 297) mRNA: GA GAACATCATTGGAATCAAT GA (SEQ ID NO: 298) AS 3′: UU CUUGUAGUAACCUUAGUUA (SEQ ID NO: 299) S 5′:    GCUAUCAUGUGUAGUAGAU UU (SEQ ID NO: 300) mRNA: TA GCTATCATGTGTAGTAGAT GC (SEQ ID NO: 301) AS 3′: UU CGAUAGUACACAUCAUCUA (SEQ ID NO: 302) S 5′:    CUGCUUAUGAUAAUGUCAA UU (SEQ ID NO: 303) mRNA: CT CTGCTTATGATAATGTCAA CA (SEQ ID NO: 304) AS 3′: UU GACGAAUACUAUUACAGUU (SEQ ID NO: 305) S 5′:    CUUGGCUUAUCCACUUUGA UU (SEQ ID NO: 306) mRNA: GT CTTGGCTTATCCACTTTGA CT (SEQ ID NO: 307) AS 3′: UU GAACCGAAUAGGUGAAACU (SEQ ID NO: 308) S 5′:    GUCUCAAAUAUUCUGUCAA UU (SEQ ID NO: 309) mRNA: AG GTCTCAAATATTCTGTCAA AC (SEQ ID NO: 310) AS 3′: UU CAGAGUUUAUAAGACAGUU (SEQ ID NO: 311) S 5′:    GCUUUCUGGUUUGAAAGAU UU (SEQ ID NO: 312) mRNA: TT GCTTTCTGGTTTGAAAGAT GC (SEQ ID NO: 313) AS 3′: UU CGAAAGACCAAACUUUCUA (SEQ ID NO: 314) S 5′:    CAACCAUCGAGCAAAUGAA UU (SEQ ID NO: 315) mRNA: AC CAACCATCGAGCAAATGAA AG (SEQ ID NO: 316) AS 3′: UU GUUGGUAGCUCGUUUACUU (SEQ ID NO: 317) S 5′:    CAUCGAGCAAAUGAAAGAU UU (SEQ ID NO: 318) mRNA: AC CATCGAGCAAATGAAAGAT GT (SEQ ID NO: 319) AS 3′: UU GUAGCUCGUUUACUUUCUA (SEQ ID NO: 320) S 5′:    GUUCAGCAUAGUACUUCAA UU (SEQ ID NO: 321) mRNA: CT GTTCAGCATAGTACTTCAA AG (SEQ ID NO: 322) AS 3′: UU CAAGUCGUAUCAUGAAGUU (SEQ ID NO: 323) S 5′:    CCACUAACUUCAUUCUAGA UU (SEQ ID NO: 324) mRNA: AA CCACTAACTTCATTCTAGA AT (SEQ ID NO: 325) AS 3′: UU GGUGAUUGAAGUAAGAUCU (SEQ ID NO: 326) S 5′:    CACCUCAGCAAUGACCAUA UU (SEQ ID NO: 327) mRNA: AA CACCTCAGCAATGACCATA TC (SEQ ID NO: 328) AS 3′: UU GUGGAGUCGUUACUGGUAU (SEQ ID NO: 329) S 5′:    CAGUGUCACUCUGUAGUUA UU (SEQ ID NO: 330) mRNA: AG CAGTGTCACTCTGTAGTTA CT (SEQ ID NO: 331) AS 3′: UU GUCACAGUGAGACAUCAAU (SEQ ID NO: 332) S 5′:    CUAGCUAUCAUGUGUAGUA UU (SEQ ID NO: 333) mRNA: CG CTAGCTATCATGTGTAGTA GA (SEQ ID NO: 334) AS 3′: UU GAUCGAUAGUACACAUCAU (SEQ ID NO: 335) S 5′:    GUAGCUUUGAGAAGCUACA UU (SEQ ID NO: 336) mRNA: CA GTAGCTTTGAGAAGCTACA TG (SEQ ID NO: 337) AS 3′: UU CAUCGAAACUCUUCGAUGU (SEQ ID NO: 338) S 5′:    GUCAAACCCUAACAAAGAA UU (SEQ ID NO: 339) mRNA: CT GTCAAACCCTAACAAAGAA GC (SEQ ID NO: 340) AS 3′: UU CAGUUUGGGAUUGUUUCUU (SEQ ID NO: 341) S 5′:    GACAUUUGGUUCUUAUCAA UU (SEQ ID NO: 342) mRNA: TG GACATTTGGTTCTTATCAA TA (SEQ ID NO: 343) AS 3′: UU CUGUAAACCAAGAAUAGUU (SEQ ID NO: 344) S 5′:    GUGUUUGAAACAUGAUACU UU (SEQ ID NO: 345) mRNA: TG GTGTTTGAAACATGATACT CC (SEQ ID NO: 346) AS 3′: UU CACAAACUUUGUACUAUGA (SEQ ID NO: 347) S 5′:    GAGAACAUCAUUGGAAUCA UU (SEQ ID NO: 348) mRNA: AT GAGAACATCATTGGAATCA AT (SEQ ID NO: 349) AS 3′: UU CUCUUGUAGUAACCUUAGU (SEQ ID NO: 350) S 5′:    GUUGUGCUGAACACAGAAA UU (SEQ ID NO: 351) mRNA: AG GTTGTGCTGAACACAGAAA TG (SEQ ID NO: 352) AS 3′: UU CAACACGACUUGUGUCUUU (SEQ ID NO: 353) S 5′:    GCUUUCUCUUCCACACAAA UU (SEQ ID NO: 354) mRNA: TT GCTTTCTCTTCCACACAAA AA (SEQ ID NO: 355) AS 3′: UU CGAAAGAGAAGGUGUGUUU (SEQ ID NO: 356) S 5′:    GUUGGUGCCUUCUUGGUAU UU (SEQ ID NO: 357) mRNA: CT GTTGGTGCCTTCTTGGTAT TG (SEQ ID NO: 358) AS 3′: UU CAACCACGGAAGAACCAUA (SEQ ID NO: 359) S 5′:    CUUGGACAUUUGGUUCUUA UU (SEQ ID NO: 360) mRNA: TT CTTGGACATTTGGTTCTTA TC (SEQ ID NO: 361) AS 3′: UU GAACCUGUAAACCAAGAAU (SEQ ID NO: 362) S 5′:    CCUGCUGAAACAUUCCAGU UU (SEQ ID NO: 363) mRNA: TT CCTGCTGAAACATTCCAGT CC (SEQ ID NO: 364) AS 3′: UU GGACGACUUUGUAAGGUCA (SEQ ID NO: 365) S 5′:    CCAGUAGCUUUGAGAAGCU UU (SEQ ID NO: 366) mRNA: AA CCAGTAGCTTTGAGAAGCT AC (SEQ ID NO: 367) AS 3′: UU GGUCAUCGAAACUCUUCGA (SEQ ID NO: 368) S 5′:    GGUCUCAAAUAUUCUGUCA UU (SEQ ID NO: 369) mRNA: TA GGTCTCAAATATTCTGTCA AA (SEQ ID NO: 370) AS 3′: UU CCAGAGUUUAUAAGACAGU (SEQ ID NO: 371) S 5′:    GAACAGAAAUGCAUUUGUA UU (SEQ ID NO: 372) mRNA: AT GAACAGAAATGCATTTGTA AT (SEQ ID NO: 373) AS 3′: UU CUUGUCUUUACGUAAACAU (SEQ ID NO: 374) S 5′:    GUCCUAACCAAGGUACCUA UU (SEQ ID NO: 375) mRNA: TG GTCCTAACCAAGGTACCTA TG (SEQ ID NO: 376) AS 3′: UU CAGGAUUGGUUCCAUGGAU (SEQ ID NO: 377) S 5′:    GCACUCAAGAAAGUUCUGA UU (SEQ ID NO: 378) mRNA: GA GCACTCAAGAAAGTTCTGA AA (SEQ ID NO: 379) AS 3′: UU CGUGAGUUCUUUCAAGACU (SEQ ID NO: 380) S 5′:    CAUGAUGGGUCAGAAACAA UU (SEQ ID NO: 381) mRNA: GA CATGATGGGTCAGAAACAA AT (SEQ ID NO: 382) AS 3′: UU GUACUACCCAGUCUUUGUU (SEQ ID NO: 383) S 5′:    CAAUGGAGAAUGGGUUAUA UU (SEQ ID NO: 384) mRNA: AG CAATGGAGAATGGGTTATA TA (SEQ ID NO: 385) AS 3′: UU GUUACCUCUUACCCAAUAU (SEQ ID NO: 386) S 5′:    CUCUAUUCUUGCCCUGAAA UU (SEQ ID NO: 387) mRNA: AT CTCTATTCTTGCCCTGAAA TA (SEQ ID NO: 388) AS 3′: UU GAGAUAAGAACGGGACUUU (SEQ ID NO: 389) S 5′:    CUUCUAUCUUCACAUUCAU UU (SEQ ID NO: 390) mRNA: AT CTTCTATCTTCACATTCAT GT (SEQ ID NO: 391) AS 3′: UU GAAGAUAGAAGUGUAAGUA (SEQ ID NO: 392) S 5′:    GUACUUCAGUGCACCUACU UU (SEQ ID NO: 393) mRNA: AT GTACTTCAGTGCACCTACT GC (SEQ ID NO: 394) AS 3′: UU CAUGAAGUCACGUGGAUGA (SEQ ID NO: 395) S 5′:    GAGUUAGAAAGGUACUUCU UU (SEQ ID NO: 396) mRNA: AT GAGTTAGAAAGGTACTTCT GT (SEQ ID NO: 397) AS 3′: UU CUCAAUCUUUCCAUGAAGA (SEQ ID NO: 398) S 5′:    GUCACUCUGUAGUUACUGU UU (SEQ ID NO: 399) mRNA: GT GTCACTCTGTAGTTACTGT GG (SEQ ID NO: 400) AS 3′: UU CAGUGAGACAUCAAUGACA (SEQ ID NO: 401) S 5′:    CACUCAAGAAAGUUCUGAA UU (SEQ ID NO: 402) mRNA: AG CACTCAAGAAAGTTCTGAA AC (SEQ ID NO: 403) AS 3′: UU GUGAGUUCUUUCAAGACUU (SEQ ID NO: 404) S 5′:    GACACAGAAAUGUGACUGU UU (SEQ ID NO: 405) mRNA: GG GACACAGAAATGTGACTGT TA (SEQ ID NO: 406) AS 3′: UU CUGUGUCUUUACACUGACA (SEQ ID NO: 407) S 5′:    CACAUGCCUACGAUUGAAA UU (SEQ ID NO: 408) mRNA: GC CACATGCCTACGATTGAAA TG (SEQ ID NO: 409) AS 3′: UU GUGUACGGAUGCUAACUUU (SEQ ID NO: 410) S 5′:    GACAUUUGGUGAGAGAAGU UU (SEQ ID NO: 411) mRNA: TC GACATTTGGTGAGAGAAGT AC (SEQ ID NO: 412) AS 3′: UU CUGUAAACCACUCUCUUCA (SEQ ID NO: 413) S 5′:    GUCCAUUGAUAUUUGGUCU UU (SEQ ID NO: 414) mRNA: AA GTCCATTGATATTTGGTCT GT (SEQ ID NO: 415) AS 3′: UU CAGGUAACUAUAAACCAGA (SEQ ID NO: 416) S 5′:    CCAUAUCCUUGGCUACUAA UU (SEQ ID NO: 417) mRNA: AA CCATATCCTTGGCTACTAA CA (SEQ ID NO: 418) AS 3′: UU GGUAUAGGAACCGAUGAUU (SEQ ID NO: 419) S 5′:    CAGCUGUAAAGUGGAAGCA UU (SEQ ID NO: 420) mRNA: GT CAGCTGTAAAGTGGAAGCA AT (SEQ ID NO: 421) AS 3′: UU GUCGACAUUUCACCUUCGU (SEQ ID NO: 422) S 5′:    GCUGAAACAUUCCAGUCCU UU (SEQ ID NO: 423) mRNA: CT GCTGAAACATTCCAGTCCT TT (SEQ ID NO: 424) AS 3′: UU CGACUUUGUAAGGUCAGGA (SEQ ID NO: 425) S 5′:    CACAAUCUUAGGUCUCAAA UU (SEQ ID NO: 426) mRNA: TT CACAATCTTAGGTCTCAAA TA (SEQ ID NO: 427) AS 3′: UU GUGUUAGAAUCCAGAGUUU (SEQ ID NO: 428) S 5′:    CUGAGUCAGACUGUCAGAA UU (SEQ ID NO: 429) mRNA: TG CTGAGTCAGACTGTCAGAA AA (SEQ ID NO: 430) AS 3′: UU GACUCAGUCUGACAGUCUU (SEQ ID NO: 431) S 5′:    GACUGUUACAGCUUUCUGU UU (SEQ ID NO: 432) mRNA: AT GACTGTTACAGCTTTCTGT GC (SEQ ID NO: 433) AS 3′: UU CUGACAAUGUCGAAAGACA (SEQ ID NO: 434) S 5′:    GUACUUCAAAGCAAGUACU UU (SEQ ID NO: 435) mRNA: TA GTACTTCAAAGCAAGTACT CA (SEQ ID NO: 436) AS 3′: UU CAUGAAGUUUCGUUCAUGA (SEQ ID NO: 437) S 5′:    CAUGUGGUAACUUGUGUUA UU (SEQ ID NO: 438) mRNA: TG CATGTGGTAACTTGTGTTA GG (SEQ ID NO: 439) AS 3′: UU GUACACCAUUGAACACAAU (SEQ ID NO: 440) S 5′:    GGAACUAUUUGCUUUCUCU UU (SEQ ID NO: 441) mRNA: TA GGAACTATTTGCTTTCTCT TC (SEQ ID NO: 442) AS 3′: UU CCUUGAUAAACGAAAGAGA (SEQ ID NO: 443) S 5′:    GAUCUUUACAAGCUCUUGA UU (SEQ ID NO: 444) mRNA: CA GATCTTTACAAGCTCTTGA AG (SEQ ID NO: 445) AS 3′: UU CUAGAAAUGUUCGAGAACU (SEQ ID NO: 446) S 5′:    CAGAUGAGAAGCUAUAACA UU (SEQ ID NO: 447) mRNA: TG CAGATGAGAAGCTATAACA GT (SEQ ID NO: 448) AS 3′: UU GUCUACUCUUCGAUAUUGU (SEQ ID NO: 449) S 5′:    CUCUGGACUUAUUGGACAA UU (SEQ ID NO: 450) mRNA: AG CTCTGGACTTATTGGACAA AA (SEQ ID NO: 451) AS 3′: UU GAGACCUGAAUAACCUGUU (SEQ ID NO: 452) S 5′:    GCUUAUGAUAAUGUCAACA UU (SEQ ID NO: 453) mRNA: CT GCTTATGATAATGTCAACA AA (SEQ ID NO: 454) AS 3′: UU CGAAUACUAUUACAGUUGU (SEQ ID NO: 455) S 5′:    GCUUUGAGAAGCUACAUGU UU (SEQ ID NO: 456) mRNA: TA GCTTTGAGAAGCTACATGT AG (SEQ ID NO: 457) AS 3′: UU CGAAACUCUUCGAUGUACA (SEQ ID NO: 458) S 5′:    CCUACUGCUUACUGUUGCU UU (SEQ ID NO: 459) mRNA: CA CCTACTGCTTACTGTTGCT TT (SEQ ID NO: 460) AS 3′: UU GGAUGACGAAUGACAACGA (SEQ ID NO: 461) S 5′:    CCUGAGGAUUUAGCAGAGA UU (SEQ ID NO: 462) mRNA: TG CCTGAGGATTTAGCAGAGA GG (SEQ ID NO: 463) AS 3′: UU GGACUCCUAAAUCGUCUCU (SEQ ID NO: 464) S 5′:    CAUAUCUGGAGCAGUAUUA UU (SEQ ID NO: 465) mRNA: CC CATATCTGGAGCAGTATTA CG (SEQ ID NO: 466) AS 3′: UU GUAUAGACCUCGUCAUAAU (SEQ ID NO: 467) S 5′:    CACAACACCUCAGCAAUGA UU (SEQ ID NO: 468) mRNA: GA CACAACACCTCAGCAATGA CC (SEQ ID NO: 469) AS 3′: UU GUGUUGUGGAGUCGUUACU (SEQ ID NO: 470) S 5′:    CUGUUGCUUUAGUCACUAA UU (SEQ ID NO: 471) mRNA: TA CTGTTGCTTTAGTCACTAA TT (SEQ ID NO: 472) AS 3′: UU GACAACGAAAUCAGUGAUU (SEQ ID NO: 473) S 5′:    CAAGAGGAUUGAAGUAGAA UU (SEQ ID NO: 474) mRNA: CA CAAGAGGATTGAAGTAGAA CA (SEQ ID NO: 475) AS 3′: UU GUUCUCCUAACUUCAUCUU (SEQ ID NO: 476) S 5′:    GAGUUGUGUUCCACGGAAA UU (SEQ ID NO: 477) mRNA: CT GAGTTGTGTTCCACGGAAA AT (SEQ ID NO: 478) AS 3′: UU CUCAACACAAGGUGCCUUU (SEQ ID NO: 479) S 5′:    CACUUGGAUUUACAUAAGA UU (SEQ ID NO: 480) mRNA: AG CACTTGGATTTACATAAGA TG (SEQ ID NO: 481) AS 3′: UU GUGAACCUAAAUGUAUUCU (SEQ ID NO: 482) S 5′:    GUGUCUGAAUGGACAGUCA UU (SEQ ID NO: 483) mRNA: GC GTGTCTGAATGGACAGTCA GG (SEQ ID NO: 484) AS 3′: UU CACAGACUUACCUGUCAGU (SEQ ID NO: 485) S 5′:    CUUGCCUUGUAUAUGGUAA UU (SEQ ID NO: 486) mRNA: TA CTTGCCTTGTATATGGTAA AG (SEQ ID NO: 487) AS 3′: UU GAACGGAACAUAUACCAUU (SEQ ID NO: 488) S 5′:    GAGAAGCUAUAACAGUGAA UU (SEQ ID NO: 489) mRNA: AT GAGAAGCTATAACAGTGAA TA (SEQ ID NO: 490) AS 3′: UU CUCUUCGAUAUUGUCACUU (SEQ ID NO: 491) S 5′:    CUCAAAGCUAGCAGAGAUA UU (SEQ ID NO: 492) mRNA: TC CTCAAAGCTAGCAGAGATA CG (SEQ ID NO: 493) AS 3′: UU GAGUUUCGAUCGUCUCUAU (SEQ ID NO: 494) S 5′:    GUGAUUUGGUUAAUCUGUA UU (SEQ ID NO: 495) mRNA: TT GTGATTTGGTTAATCTGTA TA (SEQ ID NO: 496) AS 3′: UU CACUAAACCAAUUAGACAU (SEQ ID NO: 497) S 5′:    GCUCUGGACUUAUUGGACA UU (SEQ ID NO: 498) mRNA: AA GCTCTGGACTTATTGGACA AA (SEQ ID NO: 499) AS 3′: UU CGAGACCUGAAUAACCUGU (SEQ ID NO: 500) S 5′:    CACAUACAUACGCACACAU UU (SEQ ID NO: 501) mRNA: CA CACATACATACGCACACAT GC (SEQ ID NO: 502) AS 3′: UU GUGUAUGUAUGCGUGUGUA (SEQ ID NO: 503) S 5′:    CACUUGUCAAGAAGCGUUA UU (SEQ ID NO: 504) mRNA: AA CACTTGTCAAGAAGCGTTA TG (SEQ ID NO: 505) AS 3′: UU GUGAACAGUUCUUCGCAAU (SEQ ID NO: 506) S 5′:    CUGGUUUGAAAGAUGCAGU UU (SEQ ID NO: 507) mRNA: TT CTGGTTTGAAAGATGCAGT GG (SEQ ID NO: 508) AS 3′: UU GACCAAACUUUCUACGUCA (SEQ ID NO: 509) S 5′:    GUCUCUGCUUUCUUCCUCU UU (SEQ ID NO: 510) mRNA: GC GTCTCTGCTTTCTTCCTCT GC (SEQ ID NO: 511) AS 3′: UU CAGAGACGAAAGAAGGAGA (SEQ ID NO: 512) S 5′:    CUCAGUAAAUAGCAAGUCU UU (SEQ ID NO: 513) mRNA: TA CTCAGTAAATAGCAAGTCT TT (SEQ ID NO: 514) AS 3′: UU GAGUCAUUUAUCGUUCAGA (SEQ ID NO: 515) S 5′:    GAUCUCAAGAUCUGUGACU UU (SEQ ID NO: 516) mRNA: GT GATCTCAAGATCTGTGACT TT (SEQ ID NO: 517) AS 3′: UU CUAGAGUUCUAGACACUGA (SEQ ID NO: 518) S 5′:    CAUCACAAGAAGACCUGAA UU (SEQ ID NO: 519) mRNA: CC CATCACAAGAAGACCTGAA TT (SEQ ID NO: 520) AS 3′: UU GUAGUGUUCUUCUGGACUU (SEQ ID NO: 521) S 5′:    CUCGACAUUUGGUGAGAGA UU (SEQ ID NO: 522) mRNA: CA CTCGACATTTGGTGAGAGA AG (SEQ ID NO: 523) AS 3′: UU GAGCUGUAAACCACUCUCU (SEQ ID NO: 524) S 5′:    GUAGAGGUAACCAGUAGCU UU (SEQ ID NO: 525) mRNA: GT GTAGAGGTAACCAGTAGCT TT (SEQ ID NO: 526) AS 3′: UU CAUCUCCAUUGGUCAUCGA (SEQ ID NO: 527) S 5′:    GAUAGGAUUUCUUGGACAU UU (SEQ ID NO: 528) mRNA: AA GATAGGATTTCTTGGACAT TT (SEQ ID NO: 529) AS 3′: UU CUAUCCUAAAGAACCUGUA (SEQ ID NO: 530) S 5′:    CAUGAAACCACUAACUUCA UU (SEQ ID NO: 531) mRNA: GG CATGAAACCACTAACTTCA TT (SEQ ID NO: 532) AS 3′: UU GUACUUUGGUGAUUGAAGU (SEQ ID NO: 533) S 5′:    CAUGUUCCUUUAUUCACAA UU (SEQ ID NO: 534) mRNA: GT CATGTTCCTTTATTCACAA TC (SEQ ID NO: 535) AS 3′: UU GUACAAGGAAAUAAGUGUU (SEQ ID NO: 536) S 5′:    GUUACCGGAUAACACUGAU UU (SEQ ID NO: 537) mRNA: CT GTTACCGGATAACACTGAT TA (SEQ ID NO: 538) AS 3′: UU CAAUGGCCUAUUGUGACUA (SEQ ID NO: 539) S 5′:    GCAUAGUACUUCAAAGCAA UU (SEQ ID NO: 540) mRNA: CA GCATAGTACTTCAAAGCAA GT (SEQ ID NO: 541) AS 3′: UU CGUAUCAUGAAGUUUCGUU (SEQ ID NO: 542) S 5′:    GACAUGGAAUUGGAUGACU UU (SEQ ID NO: 543) mRNA: TC GACATGGAATTGGATGACT TG (SEQ ID NO: 544) AS 3′: UU CUGUACCUUAACCUACUGA (SEQ ID NO: 545) S 5′:    GAAGAAUACUGUAUUGUGU UU (SEQ ID NO: 546) mRNA: TA GAAGAATACTGTATTGTGT GT (SEQ ID NO: 547) AS 3′: UU CUUCUUAUGACAUAACACA (SEQ ID NO: 548) S 5′:    GCUUUAGUCACUAAUUGCU UU (SEQ ID NO: 549) mRNA: TT GCTTTAGTCACTAATTGCT TT (SEQ ID NO: 550) AS 3′: UU CGAAAUCAGUGAUUAACGA (SEQ ID NO: 551) S 5′:    CACUCGACAUUUGGUGAGA UU (SEQ ID NO: 552) mRNA: TT CACTCGACATTTGGTGAGA GA (SEQ ID NO: 553) AS 3′: UU GUGAGCUGUAAACCACUCU (SEQ ID NO: 554) S 5′:    GGAUUUACAUAAGAUGAGU UU (SEQ ID NO: 555) mRNA: TT GGATTTACATAAGATGAGT TA (SEQ ID NO: 556) AS 3′: UU CCUAAAUGUAUUCUACUCA (SEQ ID NO: 557) S 5′:    GAAGUCAUAAAGAUAGGAU UU (SEQ ID NO: 558) mRNA: AA GAAGTCATAAAGATAGGAT TT (SEQ ID NO: 559) AS 3′: UU CUUCAGUAUUUCUAUCCUA (SEQ ID NO: 560) S 5′:    GGAUAACACUGAUUAGUCA UU (SEQ ID NO: 561) mRNA: CC GGATAACACTGATTAGTCA GT (SEQ ID NO: 562) AS 3′: UU CCUAUUGUGACUAAUCAGU (SEQ ID NO: 563) S 5′:    GUGUUGCUUUCCUCUGGAU UU (SEQ ID NO: 564) mRNA: CA GTGTTGCTTTCCTCTGGAT CA (SEQ ID NO: 565) AS 3′: UU CACAACGAAAGGAGACCUA (SEQ ID NO: 566) S 5′:    CUAGAUUCCAGCCAGGAUA UU (SEQ ID NO: 567) mRNA: TG CTAGATTCCAGCCAGGATA CA (SEQ ID NO: 568) AS 3′: UU GAUCUAAGGUCGGUCCUAU (SEQ ID NO: 569) S 5′:    GUGAAUAUGUGGUUUCUCU UU (SEQ ID NO: 570) mRNA: CA GTGAATATGTGGTTTCTCT TA (SEQ ID NO: 571) AS 3′: UU CACUUAUACACCAAAGAGA (SEQ ID NO: 572) S 5′:    GACAGAAUAUGUGGCCACA UU (SEQ ID NO: 573) mRNA: CT GACAGAATATGTGGCCACA CG (SEQ ID NO: 574) AS 3′: UU CUGUCUUAUACACCGGUGU (SEQ ID NO: 575) S 5′:    GAGAAGUACAAAGGUUGCA UU (SEQ ID NO: 576) mRNA: GA GAGAAGTACAAAGGTTGCA GT (SEQ ID NO: 577) AS 3′: UU CUCUUCAUGUUUCCAACGU (SEQ ID NO: 578) S 5′:    CAGAUCCAGACCAUGAUCA UU (SEQ ID NO: 579) mRNA: TG CAGATCCAGACCATGATCA CA (SEQ ID NO: 580) AS 3′: UU GUCUAGGUCUGGUACUAGU (SEQ ID NO: 581) S 5′:    CACGUAAUAUUUCAGCCAU UU (SEQ ID NO: 582) mRNA: GC CACGTAATATTTCAGCCAT TC (SEQ ID NO: 583) AS 3′: UU GUGCAUUAUAAAGUCGGUA (SEQ ID NO: 584) S 5′:    GUGAUCUCAAGAUCUGUGA UU (SEQ ID NO: 585) mRNA: CT GTGATCTCAAGATCTGTGA CT (SEQ ID NO: 586) AS 3′: UU CACUAGAGUUCUAGACACU (SEQ ID NO: 587) S 5′:    CACACUCAUUCCUUCUGCU UU (SEQ ID NO: 588) mRNA: TG CACACTCATTCCTTCTGCT CT (SEQ ID NO: 589) AS 3′: UU GUGUGAGUAAGGAAGACGA (SEQ ID NO: 590) S 5′:    CAAAGCAAGUACUCAGUAA UU (SEQ ID NO: 591) mRNA: TT CAAAGCAAGTACTCAGTAA AT (SEQ ID NO: 592) AS 3′: UU GUUUCGUUCAUGAGUCAUU (SEQ ID NO: 593) S 5′:    CAUCUUUCCAGGGAAGCAU UU (SEQ ID NO: 594) mRNA: CC CATCTTTCCAGGGAAGCAT TA (SEQ ID NO: 595) AS 3′: UU GUAGAAAGGUCCCUUCGUA (SEQ ID NO: 596) S 5′:    GAACACAGAAAUGCUCACA UU (SEQ ID NO: 597) mRNA: CT GAACACAGAAATGCTCACA GG (SEQ ID NO: 598) AS 3′: UU CUUGUGUCUUUACGAGUGU (SEQ ID NO: 599) S 5′:    CUACUAACAUCUGGAGACU UU (SEQ ID NO: 600) mRNA: GG CTACTAACATCTGGAGACT GT (SEQ ID NO: 601) AS 3′: UU GAUGAUUGUAGACCUCUGA (SEQ ID NO: 602) S 5′:    GUUCAAAUAAGCUUUCAGA UU (SEQ ID NO: 603) mRNA: AT GTTCAAATAAGCTTTCAGA CT (SEQ ID NO: 604) AS 3′: UU CAAGUUUAUUCGAAAGUCU (SEQ ID NO: 605) S 5′:    GCAAUGACCAUAUCUGCUA UU (SEQ ID NO: 606) mRNA: CA GCAATGACCATATCTGCTA TT (SEQ ID NO: 607) AS 3′: UU CGUUACUGGUAUAGACGAU (SEQ ID NO: 608) S 5′:    CUUUCUAACAGGCCCAUCU UU (SEQ ID NO: 609) mRNA: TG CTTTCTAACAGGCCCATCT TT (SEQ ID NO: 610) AS 3′: UU GAAAGAUUGUCCGGGUAGA (SEQ ID NO: 611) S 5′:    GAUUCAGUGUUGCUUUCCU UU (SEQ ID NO: 612) mRNA: AA GATTCAGTGTTGCTTTCCT CT (SEQ ID NO: 613) AS 3′: UU CUAAGUCACAACGAAAGGA (SEQ ID NO: 614) S 5′:    GGAAGCAUUAUCUUGACCA UU (SEQ ID NO: 615) mRNA: AG GGAAGCATTATCTTGACCA GC (SEQ ID NO: 616) AS 3′: UU CCUUCGUAAUAGAACUGGU (SEQ ID NO: 617) S 5′:    GUACAAAGGUUGCAGUGCU UU (SEQ ID NO: 618) mRNA: AA GTACAAAGGTTGCAGTGCT GA (SEQ ID NO: 619) AS 3′: UU CAUGUUUCCAACGUCACGA (SEQ ID NO: 620) S 5′:    CAGUAUGUUAAUACACACA UU (SEQ ID NO: 621) mRNA: AA CAGTATGTTAATACACACA TA (SEQ ID NO: 622) AS 3′: UU GUCAUACAAUUAUGUGUGU (SEQ ID NO: 623) S 5′:    GAAUGGUCCUAACCAAGGU UU (SEQ ID NO: 624) mRNA: GA GAATGGTCCTAACCAAGGT AC (SEQ ID NO: 625) AS 3′: UU CUUACCAGGAUUGGUUCCA (SEQ ID NO: 626) S 5′:    CCAUUGAUAUUUGGUCUGU UU (SEQ ID NO: 627) mRNA: GT CCATTGATATTTGGTCTGT AG (SEQ ID NO: 628) AS 3′: UU GGUAACUAUAAACCAGACA (SEQ ID NO: 629) S 5′:    CCAAUUGGCUCUAGUCACU UU (SEQ ID NO: 630) mRNA: CC CCAATTGGCTCTAGTCACT GG (SEQ ID NO: 631) AS 3′: UU GGUUAACCGAGAUCAGUGA (SEQ ID NO: 632) S 5′:    CCACGUAAUAUUUCAGCCA UU (SEQ ID NO: 633) mRNA: TG CCACGTAATATTTCAGCCA TT (SEQ ID NO: 634) AS 3′: UU GGUGCAUUAUAAAGUCGGU (SEQ ID NO: 635) S 5′:    CUUACGUCAUCCACCUUGA UU (SEQ ID NO: 636) mRNA: CT CTTACGTCATCCACCTTGA CA (SEQ ID NO: 637) AS 3′: UU GAAUGCAGUAGGUGGAACU (SEQ ID NO: 638) S 5′:    CAUGAGAACAUCAUUGGAA UU (SEQ ID NO: 639) mRNA: GA CATGAGAACATCATTGGAA TC (SEQ ID NO: 640) AS 3′: UU GUACUCUUGUAGUAACCUU (SEQ ID NO: 641) S 5′:    CUGUUCCCAAAUGCUGACU UU (SEQ ID NO: 642) mRNA: GG CTGTTCCCAAATGCTGACT CC (SEQ ID NO: 643) AS 3′: UU GACAAGGGUUUACGACUGA (SEQ ID NO: 644) S 5′:    CAACAAAGUUCGAGUAGCU UU (SEQ ID NO: 645) mRNA: GT CAACAAAGTTCGAGTAGCT AT (SEQ ID NO: 646) AS 3′: UU GUUGUUUCAAGCUCAUCGA (SEQ ID NO: 647) S 5′:    GAAGCAAUAUUACUUGCCU UU (SEQ ID NO: 648) mRNA: TG GAAGCAATATTACTTGCCT TG (SEQ ID NO: 649) AS 3′: UU CUUCGUUAUAAUGAACGGA (SEQ ID NO: 650) S 5′:    GUUCUUCAGACCUUCACCU UU (SEQ ID NO: 651) mRNA: TG GTTCTTCAGACCTTCACCT GT (SEQ ID NO: 652) AS 3′: UU CAAGAAGUCUGGAAGUGGA (SEQ ID NO: 653) S 5′:    CUCACUUUAUGAUAGGGAA UU (SEQ ID NO: 654) mRNA: AT CTCACTTTATGATAGGGAA GG (SEQ ID NO: 655) AS 3′: UU GAGUGAAAUACUAUCCCUU (SEQ ID NO: 656) S 5′:    GUUUGGAGCUCUAUCCAUA UU (SEQ ID NO: 657) mRNA: GT GTTTGGAGCTCTATCCATA TT (SEQ ID NO: 658) AS 3′: UU CAAACCUCGAGAUAGGUAU (SEQ ID NO: 659) S 5′:    CAGUAGCUUUGAGAAGCUA UU (SEQ ID NO: 660) mRNA: AC CAGTAGCTTTGAGAAGCTA CA (SEQ ID NO: 661) AS 3′: UU GUCAUCGAAACUCUUCGAU (SEQ ID NO: 662) S 5′:    GAAGUACAAAGGUUGCAGU UU (SEQ ID NO: 663) mRNA: GA GAAGTACAAAGGTTGCAGT GC (SEQ ID NO: 664) AS 3′: UU CUUCAUGUUUCCAACGUCA (SEQ ID NO: 665) S 5′:    CUUCCAGAUUUGCUCUGCA UU (SEQ ID NO: 666) mRNA: GT CTTCCAGATTTGCTCTGCA TG (SEQ ID NO: 667) AS 3′: UU GAAGGUCUAAACGAGACGU (SEQ ID NO: 668) S 5′:    CAAUAUUACUUGCCUUGUA UU (SEQ ID NO: 669) mRNA: AG CAATATTACTTGCCTTGTA TA (SEQ ID NO: 670) AS 3′: UU GUUAUAAUGAACGGAACAU (SEQ ID NO: 671) S 5′:    GAAAGAUGCAGUGGUUCCU UU (SEQ ID NO: 672) mRNA: TT GAAAGATGCAGTGGTTCCT CC (SEQ ID NO: 673) AS 3′: UU CUUUCUACGUCACCAAGGA (SEQ ID NO: 674) S 5′:    CAAUGACCAUAUCUGCUAU UU (SEQ ID NO: 675) mRNA: AG CAATGACCATATCTGCTAT TT (SEQ ID NO: 676) AS 3′: UU GUUACUGGUAUAGACGAUA (SEQ ID NO: 677) S 5′:    GAAAUACCUUGGCUGAUGU UU (SEQ ID NO: 678) mRNA: TG GAAATACCTTGGCTGATGT TG (SEQ ID NO: 679) AS 3′: UU CUUUAUGGAACCGACUACA (SEQ ID NO: 680) S 5′:    CUUGACAUGAUGGGUCAGA UU (SEQ ID NO: 681) mRNA: AC CTTGACATGATGGGTCAGA AA (SEQ ID NO: 682) AS 3′: UU GAACUGUACUACCCAGUCU (SEQ ID NO: 683) S 5′:    CUAGAAUCAUUGUAGCCAU UU (SEQ ID NO: 684) mRNA: TT CTAGAATCATTGTAGCCAT AA (SEQ ID NO: 685) AS 3′: UU GAUCUUAGUAACAUCGGUA (SEQ ID NO: 686) S 5′:    GUAACCAGUAGCUUUGAGA UU (SEQ ID NO: 687) mRNA: AG GTAACCAGTAGCTTTGAGA AG (SEQ ID NO: 688) AS 3′: UU CAUUGGUCAUCGAAACUCU (SEQ ID NO: 689) S 5′:    CUACUUCAAAUGUGGGUGU UU (SEQ ID NO: 690) mRNA: AG CTACTTCAAATGTGGGTGT TT (SEQ ID NO: 691) AS 3′: UU GAUGAAGUUUACACCCACA (SEQ ID NO: 692) S 5′:    CAUUGAUAUUUGGUCUGUA UU (SEQ ID NO: 693) mRNA: TC CATTGATATTTGGTCTGTA GG (SEQ ID NO: 694) AS 3′: UU GUAACUAUAAACCAGACAU (SEQ ID NO: 695) S 5′:    GUUUCUCUUACGUCAUCCA UU (SEQ ID NO: 696) mRNA: TG GTTTCTCTTACGTCATCCA CC (SEQ ID NO: 697) AS 3′: UU CAAAGAGAAUGCAGUAGGU (SEQ ID NO: 698) S 5′:    GUGUUAUGGAAAGAGCACA UU (SEQ ID NO: 699) mRNA: TG GTGTTATGGAAAGAGCACA GG (SEQ ID NO: 700) AS 3′: UU CACAAUACCUUUCUCGUGU (SEQ ID NO: 701) S 5′:    GGUUAUAUAAAGACUGCCU UU (SEQ ID NO: 702) mRNA: TG GGTTATATAAAGACTGCCT GC (SEQ ID NO: 703) AS 3′: UU CCAAUAUAUUUCUGACGGA (SEQ ID NO: 704) S 5′:    GGAAUUGGAUGACUUGCCU UU (SEQ ID NO: 705) mRNA: AT GGAATTGGATGACTTGCCT AA (SEQ ID NO: 706) AS 3′: UU CCUUAACCUACUGAACGGA (SEQ ID NO: 707) S 5′:    CAUUCAAGUUCGACAUGGA UU (SEQ ID NO: 708) mRNA: AC CATTCAAGTTCGACATGGA AT (SEQ ID NO: 709) AS 3′: UU GUAAGUUCAAGCUGUACCU (SEQ ID NO: 710) S 5′:    GUUUGAAAGAUGCAGUGGU UU (SEQ ID NO: 711) mRNA: TG GTTTGAAAGATGCAGTGGT TC (SEQ ID NO: 712) AS 3′: UU CAAACUUUCUACGUCACCA (SEQ ID NO: 713) S 5′:    GAAUGUUUAUGGCACCUGA UU (SEQ ID NO: 714) mRNA: TT GAATGTTTATGGCACCTGA CT (SEQ ID NO: 715) AS 3′: UU CUUACAAAUACCGUGGACU (SEQ ID NO: 716) S 5′:    CAAAUAAGCUUUCAGACUA UU (SEQ ID NO: 717) mRNA: TT CAAATAAGCTTTCAGACTA AT (SEQ ID NO: 718) AS 3′: UU GUUUAUUCGAAAGUCUGAU (SEQ ID NO: 719) S 5′:    CUAAUCAUGAGGACUCUGU UU (SEQ ID NO: 720) mRNA: AA CTAATCATGAGGACTCTGT CC (SEQ ID NO: 721) AS 3′: UU GAUUAGUACUCCUGAGACA (SEQ ID NO: 722) S 5′:    GGUAACUUGUGUUAGGGCU UU (SEQ ID NO: 723) mRNA: GT GGTAACTTGTGTTAGGGCT GT (SEQ ID NO: 724) AS 3′: UU CCAUUGAACACAAUCCCGA (SEQ ID NO: 725) S 5′:    CAUUUCAACUGUUCAGCAU UU (SEQ ID NO: 726) mRNA: TA CATTTCAACTGTTCAGCAT AG (SEQ ID NO: 727) AS 3′: UU GUAAAGUUGACAAGUCGUA (SEQ ID NO: 728) S 5′:    GAUUGAAGUAGAACAGGCU UU (SEQ ID NO: 729) mRNA: AG GATTGAAGTAGAACAGGCT CT (SEQ ID NO: 730) AS 3′: UU CUAACUUCAUCUUGUCCGA (SEQ ID NO: 731) S 5′:    CUAUUUGCUUUCUCUUCCA UU (SEQ ID NO: 732) mRNA: AA CTATTTGCTTTCTCTTCCA CA (SEQ ID NO: 733) AS 3′: UU GAUAAACGAAAGAGAAGGU (SEQ ID NO: 734) S 5′:    CUGACAGAAUAUGUGGCCA UU (SEQ ID NO: 735) mRNA: TC CTGACAGAATATGTGGCCA CA (SEQ ID NO: 736) AS 3′: UU GACUGUCUUAUACACCGGU (SEQ ID NO: 737) S 5′:    GAUUUGCUCUGCAUGUGGU UU (SEQ ID NO: 738) mRNA: CA GATTTGCTCTGCATGTGGT AA (SEQ ID NO: 739) AS 3′: UU CUAAACGAGACGUACACCA (SEQ ID NO: 740) S 5′:    GUUUCUGGUAGUUGUGGCU UU (SEQ ID NO: 741) mRNA: CG GTTTCTGGTAGTTGTGGCT TT (SEQ ID NO: 742) AS 3′: UU CAAAGACCAUCAACACCGA (SEQ ID NO: 743) S 5′:    GAAAUCCAGAGCAAGUCCU UU (SEQ ID NO: 744) mRNA: TG GAAATCCAGAGCAAGTCCT CC (SEQ ID NO: 745) AS 3′: UU CUUUAGGUCUCGUUCAGGA (SEQ ID NO: 746) S 5′:    GAUAUUUGGUCUGUAGGCU UU (SEQ ID NO: 747) mRNA: TT GATATTTGGTCTGTAGGCT GC (SEQ ID NO: 748) AS 3′: UU CUAUAAACCAGACAUCCGA (SEQ ID NO: 749) S 5′:    CAAUGACAUUAUUCGAGCA UU (SEQ ID NO: 750) mRNA: AT CAATGACATTATTCGAGCA CC (SEQ ID NO: 751) AS 3′: UU GUUACUGUAAUAAGCUCGU (SEQ ID NO: 752) S 5′:    CUUAUCCACUUUGACUCCU UU (SEQ ID NO: 753) mRNA: GG CTTATCCACTTTGACTCCT TT (SEQ ID NO: 754) AS 3′: UU GAAUAGGUGAAACUGAGGA (SEQ ID NO: 755) S 5′:    CAUAAUGUAACUGGGCAGA UU (SEQ ID NO: 756) mRNA: AA CATAATGTAACTGGGCAGA GA (SEQ ID NO: 757) AS 3′: UU GUAUUACAUUGACCCGUCU (SEQ ID NO: 758) S 5′:    CAAAUGGAAAUCCAGAGCA UU (SEQ ID NO: 759) mRNA: AA CAAATGGAAATCCAGAGCA AG (SEQ ID NO: 760) AS 3′: UU GUUUACCUUUAGGUCUCGU (SEQ ID NO: 761) S 5′:    CUUUAUGAUAGGGAAGGCU UU (SEQ ID NO: 762) mRNA: CA CTTTATGATAGGGAAGGCT AC (SEQ ID NO: 763) AS 3′: UU GAAAUACUAUCCCUUCCGA (SEQ ID NO: 764) The inhibitory nucleic acids used in the compositions and methods described herein can have any of the SEQ ID NO:3-162, 166-764 sequences, or any combination thereof. Moreover, the inhibitory nucleic acids used in the compositions and methods described herein can be complementary to any of the SEQ ID NO:3-162, 166-764 sequences, or any combination thereof. In some embodiments, the inhibitory nucleic acids used in the compositions and methods described herein include one or more of the SEQ ID NO:3-162, 166-764 sequences with one or more sequences that are complementary to any of SEQ ID NO:3-162, 166-764. For example, the inhibitory nucleic acids used in the compositions and methods described herein can include both a sense sequence selected from any of SEQ ID NO:3-162, 166-764 and the corresponding antisense sequence selected from any of SEQ ID NO:3-162, 166-764. Combinations of such nucleic acid inhibitors can also be employed in the methods and compositions described herein.

As described in more detail below the siRNAs can be expressed from an expression cassette and/or expression vector. Such an expression cassette or expression vector includes, among other things, a sequence contiguously encoding the sense and antisense siRNA sequences.

Thus, for example, when using an siRNA with the sense strand GGAACAGGTTGTTCCCAAA (SEQ ID NO:765), an expression cassette can be used that includes the SEQ ID NO:765 sequence linked to a spacer derived from an miRNA (e.g., TTCAAGAGA; SEQ ID NO:766) at the 3′ end linked to the corresponding antisense sequence (TTTGGGAACAACCTGTTCC (SEQ ID NO:767)). Thus, the non-template strand sequence used in the expression cassette for the GGAACAGGTTGTTCCCAAA (SEQ ID NO:765) siRNA will have the following sequence (SEQ ID NO:768)): GGAACAGGTTGTTCCCAAA-TTCAAGAGA-TTTGGGAACAACCTGTTCC. This sequence should be placed downstream of an RNA polymerase (RNA pol) promoter in the vector.

In some of the experiments described herein an RNA pol III promoter was used. It is known that consecutive dA residues in the template strand are required to stop transcription by RNA pol III (dTs in the non-template strand). Therefore, the vector contains six consecutive dTs in the non-template strand following the shRNA cassette. If the shRNA cassette is driven by an RNA pol II promoter, other strategies could be used to stop the transcription. Expression cassettes and/or expression vectors encoding shRNAs for all the siRNAs identified herein can be made by similar procedures.

A ribozyme is an RNA molecule with catalytic activity and is capable of cleaving a single-stranded nucleic acid such as an mRNA that has a homologous region. See, for example, Cech, Science 236: 1532-1539 (1987); Cech, Ann. Rev. Biochem. 59:543-568 (1990); Cech, Curr. Opin. Struct. Biol. 2: 605-609 (1992); Couture and Stinchcomb, Trends Genet. 12: 510-515 (1996). A ribozyme may be used to catalytically cleave an ERK2 mRNA transcript and thereby inhibit translation of the mRNA. See, for example, Haseloff et al., U.S. Pat. No. 5,641,673.

Methods of designing and constructing a ribozyme that can cleave an RNA molecule in trans in a highly sequence specific manner have been developed and described in the art. See, for example, Haseloff et al., Nature 334:585-591 (1988). A ribozyme may be targeted to a specific RNA by engineering a discrete “hybridization” region into the ribozyme. The hybridization region contains a sequence complementary to the target RNA that enables the ribozyme to specifically hybridize with the target. See, for example, Gerlach et al., EP 321,201. The target sequence may be a segment of about 5, 6, 7, 8, 9, 10, 12, 15, 20, or 50 contiguous nucleotides selected from a specific nucleotide sequence. Longer complementary sequences may be used to increase the affinity of the hybridization sequence for the target.

The hybridizing and cleavage regions of the ribozyme can be integrally related; thus, upon hybridizing to the target RNA through the complementary regions, the catalytic region of the ribozyme can cleave the target. Thus, an existing ribozyme may be modified to target an ERK2 nucleic acid of the invention by modifying the hybridization region of the ribozyme to include a sequence that is complementary to the target ERK2 nucleic acid. Alternatively, an mRNA encoding a ERK2 may be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, for example, Bartel & Szostak, Science 261:1411-1418 (1993).

The inhibitory nucleic acids of the invention may include modified nucleotides, as well as natural nucleotides such as combinations of ribose and deoxyribose nucleotides, and an antisense inhibitory nucleic acid of the invention may be of any length discussed above and that is complementary to an ERK2 mRNA.

For example the inhibitory nucleic acids can include oligonucleotides or polynucleotides containing modified backbones or non-natural internucleo side linkages. Oligonucleotides or polynucleotides having modified backbones include those that retain a phosphorus atom in the backbone, as disclosed in U.S. Pat. Nos. 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050.

Modified oligonucleotide backbones include, for example: phosphorothioates; chiral phosphorothioates; phosphorodithioates; phosphotriesters; aminoalkyl phosphotriesters; methyl and other alkyl phosphonates, including 3′-alkylene phosphonates and chiral phosphonates; phosphinates; phosphoramidates, including 3′-amino phosphoramidate and aminoalkylphosphoramidates; thionophosphoramidates; thionoalkylphosphonates; thionoalkylphosphotriesters; and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogues of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts, and free acid forms of the above modifications can also be used.

Other modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short-chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short-chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide, and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene-containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH₂ component parts, as disclosed in U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439.

Other inhibitory nucleic acids can have modifications in both the sugar and the internucleoside linkage, for example, where the backbone of the nucleotide units is replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example of such an oligonucleotide mimetic includes a peptide nucleic acid (PNA). A PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide-containing backbone, in particular an aminoethylglycine backbone. The bases are retained and are bound directly or indirectly to aza-nitrogen atoms of the amide portion of the backbone. United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262; each of which is herein incorporated by reference. Other backbone modifications which may be used in the present invention are disclosed in U.S. Pat. No. 6,303,374.

Inhibitory nucleic acid agents used in the compositions and methods described herein may also include base modifications or substitutions. As used herein, “unmodified” or “natural” bases include the purine bases adenine (A) and guanine (G) and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). “Modified” bases include but are not limited to other synthetic and natural bases, such as: 5-methylcytosine (5-me-C); 5-hydroxymethyl cytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyl uracil and cytosine; 6-azo uracil, cytosine, and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, and other 8-substituted adenines and guanines; 5-halo, particularly 5-bromo, 5-trifluoromethyl, and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Additional modified bases include those disclosed in: U.S. Pat. No. 3,687,808; Kroschwitz, J. I., ed. (1990), “The Concise Encyclopedia Of Polymer Science And Engineering,” pages 858-859, John Wiley & Sons; Englisch et al. (1991), “Angewandte Chemie,” International Edition, 30, 613; and Sanghvi, Y. S., “Antisense Research and Applications,” Chapter 15, pages 289-302, S. T. Crooke and B. Lebleu, eds., CRC Press, 1993. Such modified bases are particularly useful for increasing the binding affinity of the oligomeric compounds of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and O-6-substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S. et al. (1993), “Antisense Research and Applications,” pages 276-278, CRC Press, Boca Raton), and are presently preferred base substitutions, even more particularly when combined with 2′-O-methoxyethyl sugar modifications.

In some embodiments, expression cassettes are employed to facilitate delivery of nucleic acids that inhibit the expression of ERK2. Expression cassettes can be of any suitable construction, and can be included in any appropriate delivery vector. Such delivery vectors include plasmid DNA, viral DNA, and the like. The means by which the expression cassette in its delivery or expression vector is introduced into target cells or target organism can be transfection, reverse transfection, virus induced transfection, electroporation, direct introduction by biolystics (e.g., using a “gene gun;” BioRad, Inc., Emeryville, Calif.), and the like. Other methods that can be employed include methods widely known in the art as the methods of gene therapy. Once delivered into a target cell, or target organism the expression cassette may be maintained on an autonomously replicating piece of DNA (e.g., an expression vector), or may be integrated into the genome of the target cell or target organism.

Typically, to assemble the expression cassettes and vectors of the present invention a nucleic acid, preferably a DNA, encoding an siRNA is incorporated into a unique restriction endonuclease cleavage site, or a multiple cloning site, within a pre-existing “empty” expression cassette to form a complete recombinant expression cassette that is capable of directing the production of the siRNA transcripts of the present invention. Frequently such complete recombinant expression cassettes reside within, or inserted into, expression vectors designed for the expression of such siRNA transcripts. Methods for the construction of an expression vector for purposes of this invention should be apparent to skilled artisans apprised of the present invention. (See generally, Current Protocols in Molecular Biology, Vol. 2, Ed. Ausubel, et al., Greene Publish. Assoc. & Wiley Interscience, Ch. 13, 1988; Glover, DNA Cloning, Vol. II, IRL Press, Wash., D.C., Ch. 3, 1986; Bitter, et al., in Methods in Enzymology 153:516-544 (1987); The Molecular Biology of the Yeast Saccharomyces, Eds. Strathern et al., Cold Spring Harbor Press, Vols. I and II, 1982; and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, 1989.)

Generally, the expression cassettes inserted or assembled within the expression vectors have a promoter operably linked to a DNA encoding the siRNA that is to be employed. The promoter can be a native promoter, i.e., a promoter that is responsible for the expression of that particular gene product in cells, or it can be any other suitable promoter. Alternatively, the expression cassette can be a chimera, i.e., having a heterologous promoter that is not the native promoter responsible for the expression of the siRNA. Such heterologous promoters can even be from a different species than the target cell or organism.

The expression vector may further include an origin of DNA replication for the replication of the vectors in target cells. Preferably, the expression vectors also include a replication origin for the amplification of the vectors in, e.g., E. coli, and selection marker(s) for selecting and maintaining only those target cells harboring the expression vectors. Additionally, in some embodiments the expression vectors also contain inducible or derepressible promoters, which function to control the transcription of the siRNA transcript from the DNA that encodes it. Other regulatory sequences such as transcriptional enhancer sequences and translation regulation sequences (e.g., Shine-Dalgarno sequence) can also be operably included in the expression vectors. Transcription termination sequences, and polyadenylation signal sequences, such as those from bovine growth hormone, SV40, lacZ and AcMNPV polyhedral protein genes, may also be present.

The expression vectors of the present invention can be introduced into the target cells by any techniques known in the art, e.g., by direct DNA transformation, microinjection, electroporation, viral infection, lipofection, biolystics, and the like. The expression of the siRNA can be transient or stable, inducible or derepressible. The expression vectors can be maintained in target cells in an extrachromosomal state, i.e., as self-replicating plasmids or viruses. Alternatively, the expression vectors, or portions thereof, can be integrated into chromosomes of the target cells by conventional techniques such as site-specific recombination or selection of stable cell lines. In stable cell lines, at least the expression cassette portion of the expression vector is integrated into a chromosome of the target cells.

The vector construct can be designed to be suitable for expression in various target cells, including but not limited to bacteria, yeast cells, plant cells, nematode cells, insect cells, and mammalian and human cells. Methods for preparing expression vectors designed for expression of gene products in different target cells are well known in the art.

In some embodiments, the vector is a neurotropic adeno-associated viral vector. For example, the vector can be a neurotropic recombinant adeno-associated virus (rAAV). Adeno-associated viruses (AAV) have a linear single-stranded DNA (ssDNA) genome of approximately 4.7-kilobases (kb), with two 145 nucleotide-long inverted terminal repeats (ITR) at the termini. The virus does not encode a polymerase and therefore relies on cellular polymerases for genome replication. The ITRs flank the two viral genes—rep (replication) and cap (capsid), encoding non-structural and structural proteins, respectively.

One type of AAV vector that can be used to facilitate delivery of siRNAs into neurons and/or the brain or spinal cord is a serotype-2 rAAV vector (Musatov et al., 2002). This vector is available from Vector Biolabs (Philadelphia, Pa.). The rAAV vector-based siRNA approach presents a potent and facile tool to produce a spatial and temporal knockdown of the expression of a gene of interest (Garraway et al., 2007). Several factors indicate that the choice of a rAAV vector for the delivery of the ERK2 siRNA is a good one. First, the serotype-2 rAAV vector selectively transduces neurons in vivo (Kaspar et al., 2002). Second, rAAV is able to mediate long-term siRNA expression and gene knockdown in the transduced cells. As shown in the Examples, GFP and ERK expression was examined for 6 weeks, however, previous studies by the inventors have demonstrated that a single administration of a rAAV vector resulted in the knockdown of NR1 gene expression that persisted for at least 6 months (Garraway et al., 2007). Third, rAAV is safe and therefore convenient to use in behavioral experiments requiring repeated measurements. Fourth, rAAV mediated gene knockdown can be controlled both temporally and spatially. This conditional approach avoids embryonic lethality associated with a constitutive knock-out of ERK2 (Hatano et al., 2003; Saba-El-Leil et al., 2003; Yao et al., 2003).

Consistent with the observations described herein, several reports (Kaspar et al., 2002; South et al., 2003; Garraway et al., 2007) have provided evidence at the ultrastructural and light microscope levels as well as direct behavioral threshold evidence that the injection of AAV into the brain or spinal cord dorsal horn does not result in significant immune or glial activation or behavioral sensitization. It has been suggested that a high dose of a siRNA might induce nonspecific and off-target effects (Bridge et al., 2003; Sledz et al., 2003). However, previous studies by the inventors indicated that neither an NR1 siRNA nor a control siRNA delivered by the rAAV vector induced detectable cellular toxicity (Garraway et al., 2007). Transduced neurons exhibited unaltered expression of NeuN compared with the contralateral side. In addition, no signs of gliosis or neuronal damage were observed in experiments described herein (FIG. 2G).

Moreover, the specificity of the rAAV vectors employed herein not only for neuronal tissues but also for ERK2 is clearly demonstrated in the Examples. The vectors expressed different siRNAs but induced a similar degree of marker GFP expression and knockdown of the ERK2 mRNA in the spinal cord dorsal horn. Thus, this knockdown is specific to the targeted tissues. Moreover, the knockdown by the siRNAs described herein (Examples 1-3) clearly targets the ERK2mRNA and protein as revealed by in situ hybridization and Western blot. However, the closely related ERK1 was unaffected at the protein level. Thus, the utility of the siRNAs for specifically reducing ERK2 expression in neuronal tissues is demonstrated.

ERK2 Compound Inhibitors

In some embodiments, the ERK2 inhibitor is a compound or small molecule. Such compounds are readily available, for example, are described in U.S. Pat. Nos. 7,345,054, 7,304,061, 7,253,187 and 6,743,791, which are specifically incorporated herein by reference in their entireties.

Compounds that can be used in the compositions and methods described herein include, for example, compounds of formula I:

or a pharmaceutically acceptable salt thereof, wherein:

A¹ is N or C¹⁰;

A² is N or CR¹¹;

T is selected from —C(R⁷)₂—, C(O)—, —C(O)C(O)—, —C(O)NR⁷—, —C(O)NR⁷NR⁷—, —CO₂—, —OC(O)—, —NR⁷CO₂—, —O—, —NR⁷C(O)NR⁷—, OC(O)NR⁷—, —NR⁷NR⁷—, —NR⁷C(O)—, —S—, —SO—, —SO₂—NR⁷—, —SO₂NR⁷—, —NR⁷SO₂—, —NR⁷O₂—, or —NR⁷SO₂NR⁷—;

m is selected from zero or one;

R¹ is selected from: (a) hydrogen, CN, halogen, R, N(R⁷)₂, OR, or OH, wherein m is zero; or (b) hydrogen or R, wherein m is one;

X is selected from —C(O)—, —C(O)NR⁷—, —NR⁷C(O)—, —NR⁷SO₂—, —SO₂NR⁷—, —S(O)—, or —SO₂—;

R² is selected from —(CH₂)_(y)R⁵, —(CH₂)_(y)CH(R⁵)₂, —(CH₂)_(y)CH(R⁸)(R⁵), —(CH₂)_(y)CH(R⁸)CH(R⁵)₂, —N(R⁴)₂, —NR⁴(CH₂)_(y)N(R⁴)₂, —ON(R⁷)₂, or —NR⁷OR⁶;

y is 0-6;

R³ is selected from —R, —OR⁶, —SR⁶, —S(O)R⁶, —SO₂R⁶, —ON(R⁷)₂, —N(R)₂, —NRN(R⁷)₂, or —NROR⁶;

R⁶ is selected from hydrogen or —R;

each R is independently selected from an optionally substituted group selected from C₁₋₆ aliphatic; 3-7 membered saturated, partially saturated, or aromatic monocyclic ring having zero to three heteroatoms independently selected from nitrogen, sulfur, or oxygen; or an 8-10 membered saturated, partially saturated, or aromatic bicyclic ring having zero to four heteroatoms independently selected from nitrogen, sulfur, or oxygen;

each R⁴ is independently selected from —R, —R⁷, —COR⁷, —CO₂R, —CON(R⁷)₂, —SO₂R⁷, —(CH₂)_(y)R⁵, or —(CH₂)_(y)CH(R⁵)₂;

each R⁵ is independently selected from —R, —OR, —CO₂R, —(CH₂)_(y)N(R⁷)₂, —N(R⁷)₂, —OR⁷, —SR⁷, —NR⁷C(O)R⁷, —NR⁷CON(R⁷)₂, —C(O)N(R⁷)₂, —SO₂R⁷, —NR⁷SO₂R⁷, —C(O)R⁷, —CN, or —SO₂N(R⁷)₂;

each R⁷ is independently selected from hydrogen or an optionally substituted C₁₋₆ aliphatic group, or two R⁷ groups bound to the same nitrogen are taken together with the nitrogen to form a 3-7 membered heterocyclic ring having 0-2 heteroatoms in addition to the nitrogen, independently selected from nitrogen, oxygen, or sulfur;

R⁸ is selected from —R, —(CH₂)_(w)OR⁷, —(CH₂)_(w)N(R⁴)₂, or —(CH₂)_(w)SR⁷;

each w is independently selected from 0-4;

R⁹ is selected from hydrogen, a C₁₋₆ aliphatic group, C(O)R⁷, C(O)OR⁷, or SO₂R⁷;

R¹⁰ is selected from R⁷, halogen, CN, NO₂, OR⁷, SR⁷, N(R⁷)₂, C(O)R⁷, or CO₂R⁷; or R¹⁰ and R³ are taken together to form an optionally substituted 5-7 membered saturated, partially saturated, or aromatic ring having 0-2 heteroatoms independently selected from nitrogen, oxygen, and sulfur;

R¹¹ is selected from R⁷, halogen, CN, NO₂, OR⁷, SR⁷, N(R⁷)₂, C(O)R⁷, or CO₂R⁷;

R¹² is selected from R⁷, CN, NO₂, halogen, N(R⁷)₂, SR⁷, and OR⁷; and

R¹³ is selected from R⁷, CN, NO₂, halogen, N(R⁷)₂, SR⁷, and OR⁷;

provided that only one of R¹² and R¹³ is a 3-7 membered saturated, partially saturated, or aromatic monocyclic ring having zero to three heteroatoms independently selected from nitrogen, sulfur, or oxygen; or an 8-10 membered saturated, partially saturated, or aromatic bicyclic ring having zero to four hetero atoms independently selected from nitrogen, sulfur, or oxygen.

The phrase “optionally substituted” is used interchangeably with the phrase “substituted or unsubstituted.” Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and each substitution is independent of the other.

The term “aliphatic” or “aliphatic group” as used herein means a straight-chain or branched C₁-C₁₂ hydrocarbon chain that is completely saturated or that contains one or more units of unsaturation, or a monocyclic C₃-C₈ hydrocarbon or bicyclic C₈-C₁₂ hydrocarbon that is completely saturated or that contains one or more units of unsaturation, but which is not aromatic (also referred to herein as “carbocycle” or “cycloalkyl”), that has a single point of attachment to the rest of the molecule wherein any individual ring in said bicyclic ring system has 3-7 members. For example, suitable aliphatic groups include, but are not limited to, linear or branched or alkyl, alkenyl, alkynyl groups and hybrids thereof such as (cycloalkyl)alkyl, (cycloalkenyl)alkyl or (cycloalkyl)alkenyl.

The terms “alkyl,” “alkoxy”, “hydroxyalkyl,” “alkoxyalkyl” and “alkoxycarbonyl,” used alone or as part of a larger moiety includes both straight and branched chains containing one to twelve carbon atoms. The terms “alkenyl” and “alkynyl” used alone or as part of a larger moiety shall include both straight and branched chains containing two to twelve carbon atoms.

The terms “haloalkyl,” haloalkenyl and “haloalkoxy” means alkyl, alkenyl or alkoxy, as the case may be, substituted with one or more halogen atoms. The term “halo” or “halogen” means F, Cl, Br, or I.

The term “heteroatom” means nitrogen, oxygen, or sulfur and includes any oxidized form of nitrogen and sulfur, and the quaternized form of any basic nitrogen. Also the term “nitrogen” includes a substitutable nitrogen of a heterocyclic ring. As an example, in a saturated or partially unsaturated ring having 0-3 heteroatoms selected from oxygen, sulfur or nitrogen, the nitrogen may be N (as in 3,4-dihydro-2H-pyrrolyl), NH (as in pyrrolidinyl) or NR⁺ (as in N-substituted pyrrolidinyl).

The term “aryl” used alone or as part of a larger moiety as in “aralkyl,” “aralkoxy” or “aryloxyalkyl,” refers to monocyclic, bicyclic and tricyclic carbocyclic ring systems having a total of five to fourteen ring members, wherein at least one ring in the system is aromatic and wherein each ring in the system contains 3 to 8 ring members. The term “aryl” may be used interchangeably with the term “aryl ring.”

The term “heterocycle,” “heterocyclyl” or “heterocyclic” as used herein means monocyclic, bicyclic or tricyclic ring systems having five to fourteen ring members in which one or more ring members is a heteroatom, wherein each ring in the system contains 3 to 7 ring members and is non-aromatic.

The term “heteroaryl,” used alone or as part of a larger moiety as in “heteroaralkyl” or “heteroarylalkoxy” refers to monocyclic, bicyclic and tricyclic ring systems having a total of five to fourteen ring members, and wherein: 1) at least one ring in the system is aromatic; 2) at least one ring in the system contains one or more heteroatoms; and 3) each ring in the system contains 3 to 7 ring members. The term “heteroaryl” may be used interchangeably with the term “heteroaryl ring” or the term “hetero aromatic.”

An aryl (including aralkyl, aralkoxy, aryloxyalkyl and the like) or heteroaryl (including heteroaralkyl, heteroarylalkoxy and the like) group may contain one or more substituents. Substituents on the unsaturated carbon atom of an aryl, heteroaryl, aralkyl, or heteroaralkyl group are selected from halogen; haloalkyl; —CF₃; —R^(∘); —OR^(∘); —SR^(∘); 1,2-methylene-dioxy; 1,2-ethylenedioxy; dimethyleneoxy; protected OH (such as acyloxy); phenyl (Ph); Ph substituted with R.sup.o; —O(Ph); —O-(Ph) substituted with R^(∘); —CH₂(Ph); —CH₂(Ph) substituted with R^(∘); —CH₂CH₂(Ph); —CH₂CH₂(Ph) substituted with R^(∘); —NO₂; —CN; —N(R^(∘))₂; —NR^(∘)C(O)R^(∘); —NR^(∘)C(O)N(R^(∘))₂; —NR^(∘)CO₂R^(∘); —NR^(∘)NR^(∘)C(O)R^(∘); —NR^(∘)NR^(∘)C(O)N(R^(∘))₂; —NR^(∘)NR^(∘)CO₂R^(∘); —C(O)C(O)R^(∘); —C(O)CH₂C(O)R^(∘); —CO₂R^(∘); —C(O)R^(∘); —C(O)N(R^(∘))₂; —OC(O)N(R^(∘))₂; —S(O)₂R^(∘); —SO₂N(R^(∘))₂; —S(O)R^(∘); —NR^(∘)SO₂N(R^(∘))₂; —NR^(∘)SO₂R^(∘); —C(═S)N(R^(∘))₂; —C(═NH)—N(R^(∘))₂; —(CH₂)_(y)NHC(O)R^(∘); —(CH₂)_(y)R^(∘); —(CH₂)_(y)NHC(O)NHR^(∘); —(CH₂)_(y)NHC(O)OR^(∘); —(CH₂)_(y)NHS(O)R^(∘); —(CH₂)_(y)NHSO₂R^(∘); or))—(CH₂)_(y)NHC(O)CH(V_(z)—R^(∘))(R^(∘)), wherein each R^(∘)is independently selected from hydrogen, optionally substituted C₁₋₆ aliphatic, an unsubstituted 5-6 membered heteroaryl or heterocyclic ring, phenyl (Ph), —O(Ph), or —CH₂(Ph)-CH₂(Ph), wherein y is 0-6; z is 0-1; and V is a linker group.

When R^(∘)is C₁₋₆ aliphatic, it is optionally substituted with one or more substituents selected from —NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄ aliphatic)₂, —S(O)(C₁₋₄ aliphatic), —SO₂(C₁₋₄ aliphatic), halogen, —(C₁₋₄ aliphatic), —OH, —O—(C₁₋₄ aliphatic), —NO₂, —CN, —CO₂H, —CO₂(C₁₋₄ aliphatic), —O(halo C₁₋₄ aliphatic), or -halo(C₁₋₄ aliphatic); wherein each C₁₋₄ aliphatic is unsubstituted.

An aliphatic group or a non-aromatic heterocyclic ring may contain one or more substituents. Substituents on the saturated carbon of an aliphatic group or of a non-aromatic heterocyclic ring are selected from those listed above for the unsaturated carbon of an aryl or heteroaryl group and the following: ═O, ═S, ═NN(R*)₂, ═N—, ═NNHC(O)R*, ═NNHCO₂(alkyl), ═NNHSO₂(alkyl), or ═NR*, where each R* is independently selected from hydrogen or an optionally substituted C₁₋₆ aliphatic. When R* is C₁₋₆ aliphatic, it is optionally substituted with one or more substituents selected from —NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄ aliphatic)₂, halogen, —OH, —O—(C₁₋₄ aliphatic), —NO₂, —CN, —CO₂H, —CO₂(C₁₋₄ aliphatic), —O(halo C₁₋₄ aliphatic), or -halo(C₁₋₄ aliphatic); wherein each C₁₋₄ aliphatic is unsubstituted.

Substituents on the nitrogen of a non-aromatic heterocyclic ring are selected from —R⁺, —N(R⁺)₂, —C(O)R⁺, —CO₂R⁺, —C(O)C(O)R⁺, —C(O)CH₂C(O)R⁺, —SO₂R+, —SO₂N(R⁺)₂, —C(═S)N(R⁺)₂, —C(═NH)—N(R⁺)₂, or —NR⁺SO₂R⁺; wherein each R⁺ is independently selected from hydrogen, an optionally substituted C₁₋₆ aliphatic, optionally substituted phenyl (Ph), optionally substituted —O(Ph), optionally substituted —CH₂(Ph), optionally substituted —CH₂CH₂(Ph), or an unsubstituted 5-6 membered heteroaryl or heterocyclic ring. When R⁺ is a C₁₋₆ aliphatic group or a phenyl ring, it is optionally substituted with one or more substituents selected from —NH₂, —NH(C₁₋₄ aliphatic), —N(C₁₋₄ aliphatic)₂, halogen, —(C₁₋₄ aliphatic), —OH, —O—(C₁₋₄ aliphatic), —NO₂, —CN, —CO₂H, —CO₂(C₁₋₄ aliphatic), —O(halo C₁₋₄ aliphatic), or -halo(C₁₋₄ aliphatic); wherein each C₁₋₄ aliphatic is unsubstituted.

The V linker group refers to an organic moiety that connects two parts of a compound. For example, V linkers are comprised of —O—, —S—, —NR*—, —C(R*)₂—, —C(O)—, or an alkylidene chain. The alkylidene chain is a saturated or unsaturated, straight or branched, C₁₋₆ carbon chain which is optionally substituted, and wherein up to two non-adjacent saturated carbons of the chain are optionally replaced by —C(O)—, —C(O)C(O)—, —C(O)NR*—, —C(O)NR*NR*—, —CO₂—, —OC(O)—, —NR*CO₂—, —O—, —NR*C(O)NR*—, —OC(O)NR*—, —NR*NR*—, —NR*C(O)—, —S—, —SO—, —SO₂—, —NR*—, —SO₂NR*—, or —NR*SO₂—; wherein R* is selected from hydrogen or C₁₋₄ aliphatic. Optional substituents on the alkylidene chain are as described above for an aliphatic group.

Unless otherwise stated, structures depicted herein are also meant to include all stereochemical forms of the structure; i.e., the R and S configurations for each asymmetric center. Therefore, single stereochemical isomers as well as enantiomeric and diastereomeric mixtures of the present compounds are within the scope of the invention. Unless otherwise stated, structures depicted herein are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of a hydrogen by a deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon are within the scope of this invention.

Compounds of this invention may exist in alternative tautomeric forms. Unless otherwise indicated, the representation of either tautomer is meant to include the other.

Examples of useful ERK2 inhibitor compounds include the following:

Antibodies

Another agent that can be used to inhibit ERK2 is an antibody preparation. Such anti-ERK2 antibodies can be used in the compositions and methods described herein. The term “antibody,” as used herein, refers to a full-length immunoglobulin molecule or an immunologically-active fragment of an immunoglobulin molecule such as the Fab or F(ab′)₂ fragment generated by, for example, cleavage of the antibody with an enzyme such as pepsin or co-expression of an antibody light chain and an antibody heavy chain in bacteria, yeast, insect cell or mammalian cell. The antibody can also be an IgG, IgD, IgA, IgE or IgM antibody.

As used herein, the term “binds specifically” or “specifically binds,” in reference to an antibody/antigen interaction, means that the antibody binds with a particular antigen (e.g., ERK2) without substantially binding to other unrelated antigens. For example, in some embodiments, the anti-ERK2 antibodies bind with greater affinity to ERK2 than to ERK1. Thus, the anti-ERK2 antibodies can have at least 50% or greater affinity, or greater affinity, to ERK2 than to ERK1. In addition, the anti-ERK2 antibodies can have about 75% or greater affinity, and more preferably, about 90% or greater affinity, to ERK2 than to other unrelated polypeptides.

An antibody directed against ERK2 can be a polyclonal or monoclonal antibody. Polyclonal antibodies can be obtained by immunizing a mammal with a mutant polypeptide of the invention, and then isolating antibodies from the blood of the mammal using standard techniques. The antibodies can be evaluated for affinity to ERK2 using standard procedures including, for example, enzyme linked immunosorbent assay (ELISA) to determine antibody titer and protein A chromatography to obtain the antibody-containing an IgG fraction.

The anti-ERK2 antibodies can be monoclonal or polyclonal antibodies. A monoclonal antibody is a population of molecules having a common antigen binding site that binds specifically with a particular antigenic epitope. A monoclonal antibody can be obtained by selecting an antibody-producing cell from a mammal that has been immunized with ERK2, and fusing the antibody-producing cell, e.g. a B cell, with a myeloma to generate an antibody-producing hybridoma. A monoclonal antibody can also be obtained by screening a recombinant combinatorial library such as an antibody phage display library. See, for example, PHAGE DISPLAY—A LABORATORY MANUAL, Barbas, et al., eds. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001; and Kontermann & Dübel, ANTIBODY ENGINEERING, Heidelberg: Springer-Verlag. Berlin, 2001.

An anti-ERK2 antibody can also be a murine, chimeric, humanized or fully human antibody. A murine antibody is an antibody derived entirely from a murine source, for example, an antibody derived from a murine hybridoma generated from the fusion of a mouse myeloma cell and a mouse B-lymphocyte cell. A chimeric antibody is an antibody that has variable regions derived from a non-human source, e.g. murine or primate, and constant regions derived from a human source. A humanized antibody has antigen-binding regions, e.g. complementarity-determining regions, derived from a mouse source, and the remaining variable regions and constant regions derived from a human source. A fully human antibody is antibody from human cells or derived from transgenic mice carrying human antibody genes.

Methods to generate antibodies are well known in the art. For example, a polyclonal antibody of the invention can be prepared by immunizing a suitable animal with ERK2. The animal can be, for example, a rabbit, goat, sheep, rabbit, hamster, chicken, cow, or mouse. At the appropriate time after immunization, antibody molecules can be isolated from the animal, e.g. from the blood or other fluid of the animal, and further purified using standard techniques that include, without limitation, precipitation using ammonium sulfate, gel filtration chromatography, ion exchange chromatography or affinity chromatography using protein A. In addition, an antibody-producing cell of the mammal can be isolated and used to prepare a hybridoma cell that secretes a monoclonal antibody of the invention. Techniques for preparing monoclonal antibody-secreting hybridoma cells are available in the art. See, for example, Kohler and Milstein, Nature 256:495-97 (1975) and Kozbor et al. Immunol Today 4: 72 (1983). A monoclonal antibody against ERK2 can also be prepared using other methods available in the art, such as, for example, expression from a recombinant DNA molecule, or screening of a recombinant combinatorial immunoglobulin library using a mutant polypeptide of the invention.

Methods to generate chimeric and humanized monoclonal antibodies are also readily available in the art and include, for example, methods involving recombinant DNA technology. A chimeric antibody can be produced by expression from a nucleic acid that encodes a non-human variable region and a human constant region of an antibody molecule. See, for example, Morrison et al., Proc. Nat. Acad. Sci. U.S.A. 86: 6851 (1984). A humanized antibody can be produced by expression from a nucleic acid that encodes non-human antigen-binding regions (complementarity-determining regions) and a human variable region (without antigen-binding regions) and human constant regions. See, for example, Jones et al., Nature 321:522-24 (1986); and Verhoeven et al., Science 239:1534-36 (1988). Completely human antibodies can be produced by immunizing engineered transgenic mice that express only human heavy and light chain genes. In this case, therapeutically useful monoclonal antibodies can then be obtained using conventional hybridoma technology. See, for example, Lonberg & Huszar, Int. Rev. Immunol. 13:65-93 (1995). Nucleic acids and techniques involved in design and production of antibodies are well known in the art. See, for example, Batra et al., Hybridoma 13:87-97 (1994); Berdoz et al., PCR Methods Appl. 4: 256-64 (1995); Boulianne et al. Nature 312:643-46 (1984); Carson et al., Adv. Immunol. 38:274-311 (1986); Chiang et al., Biotechniques 7:360-66 (1989); Cole et al., Mol. Cell. Biochem. 62:109-20 (1984); Jones et al., Nature 321: 522-25 (1986); Larrick et al., Biochem Biophys. Res. Commun. 160:1250-56 (1989); Morrison, Annu. Rev. Immunol. 10:239-65 (1992); Morrison et al., Proc. Nat'l Acad. Sci. USA 81: 6851-55 (1984); Orlandi et al., Pro. Nat'l Acad. Sci. U.S.A. 86:3833-37 (1989); Sandhu, Crit. Rev. Biotechnol. 12:437-62 (1992); Gavilondo & Larrick, Biotechniques 29: 128-32 (2000); Huston & George, Hum. Antibodies. 10:127-42 (2001); Kipriyanov & Le Gall, Mol. Biotechnol. 26: 39-60 (2004).

Another method for generating antibodies involves a Selected Lymphocyte Antibody Method (SLAM). The SLAM technology permits the generation, isolation and manipulation of monoclonal antibodies without needing to generate a hybridoma. The methodology principally involves the growth of antibody forming cells, the physical selection of specifically selected antibody forming cells, the isolation of the genes encoding the antibody and the subsequent cloning and expression of those genes.

The nucleic acids encoding the antibodies can be mutated to optimize the affinity, selectivity, binding strength or other desirable property of an antibody. A mutant antibody refers to an amino acid sequence variant of an antibody. In general, one or more of the amino acid residues in the mutant antibody is different from what is present in the reference antibody. Such mutant antibodies necessarily have less than 100% sequence identity or similarity with the reference amino acid sequence. In general, mutant antibodies have at least 75% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the reference antibody. Preferably, mutant antibodies have at least 80%, more preferably at least 85%, even more preferably at least 90%, and most preferably at least 95% amino acid sequence identity or similarity with the amino acid sequence of either the heavy or light chain variable domain of the reference antibody.

The antibodies can be isolated antibodies. An isolated antibody is one that has been identified and separated and/or recovered from a component of the environment in which it was produced. Contaminant components of its production environment are materials that would interfere with diagnostic or therapeutic uses for the antibody, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. The term “isolated antibody” also includes antibodies within recombinant cells because at least one component of the antibody's natural environment will not be present. Ordinarily, however, isolated antibody will be prepared by at least one purification step.

If desired, the anti-ERK2 antibodies can be purified by any available procedure. For example, the antibodies can be affinity purified by binding an antibody preparation to a solid support to which the antigen used to raise the antibodies is bound. After washing off contaminants, the antibody can be eluted by known procedures. Those of skill in the art are cognizant of various techniques common in the immunology arts for purification and/or concentration of polyclonal antibodies, as well as monoclonal antibodies (see for example, Coligan, et al., Unit 9, Current Protocols in Immunology, Wiley Interscience, 1991, incorporated by reference).

Monoclonal antibodies can be isolated and purified from hybridoma cultures by a variety of well-established techniques. Such isolation techniques include affinity chromatography with Protein-A Sepharose, size-exclusion chromatography, and ion-exchange chromatography. See, e.g., Coligan, et al., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al., Purification of Immunoglobulin G (IgG), in: METHODS IN MOLECULAR BIOLOGY, Vol. 10, pages 79-104 (Humana Press (1992).

In some embodiments, the antibody will be purified as measurable by at least three different methods: 1) to greater than 95% by weight of antibody as determined by the Lowry method, and preferably more than 99% by weight; 2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence by use of a spinning cup sequenator; or 3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions using Coomassie blue or, preferably, silver stain.

The antibodies described herein include immunologically-active fragments of antibodies. Methods of making antibody fragments are also known in the art (see for example, Harlow and Lane, ANTIBODIES: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, New York, (1988), incorporated herein by reference). Antibody fragments of the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab′)2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab′ fragments and an Fc fragment directly. These methods are described, for example, in U.S. Pat. No. 4,036,945 and U.S. Pat. No. 4,331,647, and references contained therein. These patents are hereby incorporated in their entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light-heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody. For example, Fv fragments comprise an association of V_(H) and V_(L) chains. This association may be noncovalent or the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise V_(H) and V_(L) chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the V_(H) and V_(L) domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by Whitlow, et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 97 (1991); Bird, et al., Science 242:423-426 (1988); Ladner, et al, U.S. Pat. No. 4,946,778; and Pack, et al., Bio/Technology 11:1271-77 (1993).

Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides (“minimal recognition units”) can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2, page 106 (1991). The antibodies described herein can be any CDR-containing polypeptides.

Compositions

The invention also relates to compositions containing a nucleic acid that inhibits expression of ERK2 protein with SEQ ID NO:1 (or an expression cassette or vector that encodes such a nucleic acid), a compound that can inhibit ERK2 activity or an anti-ERK2 antibody can bind with specificity to a polypeptide having SEQ ID NO:1. The compositions can also contain a carrier, for example, a pharmaceutically acceptable carrier.

By “pharmaceutically acceptable” it is meant a carrier, diluent, excipient, and/or salt that is compatible with the other ingredients of the formulation, and not deleterious to the recipient thereof.

In some embodiments, the therapeutic agents of the invention (e.g., a nucleic acid that inhibits ERK2 expression, a vector encoding such a nucleic acid, a compound that inhibits ERK2 activity and/or an anti-ERK2 antibody), are administered in a “therapeutically effective amount.” Such a therapeutically effective amount is an amount sufficient to obtain the desired physiological effect, e.g., treatment of a condition, disorder, disease and the like or reduction in symptoms of the condition, disorder, disease and the like. For example, the therapeutic agents can be administered to treat a condition, disorder, or disease that involves acute or chronic pain.

To achieve the desired effect(s), the nucleic acid that inhibits ERK2 expression, the vector encoding such a nucleic acid, the compound that inhibits ERK2 activity, the anti-ERK2 antibody and combinations thereof, may be administered as single or divided dosages. For example, nucleic acids, vectors, compounds and/or antibodies can be administered in dosages of at least about 0.01 mg/kg to about 500 to 750 mg/kg, of at least about 0.01 mg/kg to about 300 to 500 mg/kg, at least about 0.1 mg/kg to about 100 to 300 mg/kg or at least about 1 mg/kg to about 50 to 100 mg/kg of body weight, although other dosages may provide beneficial results. The amount administered will vary depending on various factors including, but not limited to, the nucleic acid, vector, compound or antibody chosen for administration, the disease, the weight, the physical condition, the health, the age of the mammal, and if the nucleic acid, vector, compound or antibody is chemically modified. Such factors can be readily determined by the clinician employing animal models or other test systems that are available in the art.

Administration of the therapeutic agents in accordance with the present invention may be in a single dose, in multiple doses, in a continuous or intermittent manner, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic or prophylactic, and other factors known to skilled practitioners. The administration of the therapeutic agents and compositions of the invention may be essentially continuous over a preselected period of time or may be in a series of spaced doses. Both local and systemic administration is contemplated.

To prepare the composition, nucleic acids, vectors, compounds, antibodies and other agents are synthesized or otherwise obtained, purified as necessary or desired and then lyophilized and stabilized. These agents can then be adjusted to the appropriate concentration, and optionally combined with other agents. The absolute weight of a given nucleic acid, vector, compound, antibody and/or other agent included in a unit dose can vary widely. For example, about 0.01 to about 2 g, or about 0.1 to about 500 mg, of at least one nucleic acid, vector, compound or antibody of the invention, or a plurality or combination of nucleic acids, vectors, compounds and/or antibodies can be administered. Alternatively, the unit dosage can vary from about 0.01 g to about 50 g, from about 0.01 g to about 35 g, from about 0.1 g to about 25 g, from about 0.5 g to about 12 g, from about 0.5 g to about 8 g, from about 0.5 g to about 4 g, or from about 0.5 g to about 2 g.

Daily doses of the therapeutic agents of the invention can vary as well. Such daily doses can range, for example, from about 0.1 g/day to about 50 g/day, from about 0.1 g/day to about 25 g/day, from about 0.1 g/day to about 12 g/day, from about 0.5 g/day to about 8 g/day, from about 0.5 g/day to about 4 g/day, and from about 0.5 g/day to about 2 g/day.

Thus, one or more suitable unit dosage forms comprising the nucleic acids, vectors, compounds and/or anti-ERK2 antibodies can be administered by a variety of routes including parenteral (including subcutaneous, intravenous, intramuscular and intraperitoneal), oral, rectal, dermal, transdermal, intrathoracic, intrapulmonary and intranasal (respiratory) routes. The nucleic acids, vectors, compounds and/or antibodies may also be formulated for sustained release (for example, using microencapsulation, see WO 94/07529, and U.S. Pat. No. 4,962,091). The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known to the pharmaceutical arts. Such methods may include the step of mixing the therapeutic agent with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, introducing or shaping the product into the desired delivery system.

The compositions of the invention may be prepared in many forms that include aqueous solutions, suspensions, tablets, hard or soft gelatin capsules, and liposomes and other slow-release formulations, such as shaped polymeric gels. However, administration of compounds, nucleic acids, vectors and/or antibodies often involves parenteral or local administration of the nucleic acids, vectors, compounds and/or antibodies in an aqueous solution or sustained release vehicle.

Thus while the nucleic acids, vectors, compounds and/or antibodies may sometimes be administered in an oral dosage form, that oral dosage form is typically formulated such that the protein, nucleic acid or antibody is released into the intestine after passing through the stomach. Such formulations are described in U.S. Pat. No. 6,306,434 and in the references contained therein.

Liquid pharmaceutical compositions may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, dry powders for constitution with water or other suitable vehicle before use. Such liquid pharmaceutical compositions may contain conventional additives such as suspending agents, emulsifying agents, non-aqueous vehicles (which may include edible oils), or preservatives.

A protein, nucleic acid, compound or antibody can be formulated for parenteral administration (e.g., by injection, for example, bolus injection or continuous infusion) and may be presented in unit dosage form in ampoules, prefilled syringes, small volume infusion containers or multi-dose containers with an added preservative. The pharmaceutical compositions may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Suitable carriers include saline solution and other materials commonly used in the art.

The compositions can also contain other ingredients such as other analgesics (e.g., acetaminophen, ibuprofen, salicylic acid), vitamins, anti-microbial agents, or preservatives. It will be appreciated that the amount of an nucleic acid, vector, compound or antibody required for use in treatment will vary not only with the particular carrier selected but also with the route of administration, the nature of the condition being treated and the age and condition of the patient. Ultimately the attendant health care provider may determine proper dosage. In addition, a pharmaceutical composition may be formulated as a single unit dosage form.

The following non-limiting Examples illustrate certain aspects of the invention.

Example 1 Materials and Methods

This Example illustrates some of the materials and methods used to determine that ERK2 inhibitors reduce pain in animals.

Experimental Animals and Drugs.

Adult male C57BL/6 mice (Jackson Labs) weighing 20-30 grams were used for this study. Experiments were performed in accordance with National Institute of Health Guidelines for the Care and Use of Laboratory Animals. The experimental protocol (#0508-392A) was approved by the Institutional Animal Care and Use Committee at Weill Cornell Medical College. Animals were housed under 12 hour light/dark cycles in a pathogen-free room with free access to water and food.

Design and Screening of siRNAs and Viral Vector Production.

The approaches used to design and screen for siRNAs targeting the expression of a single gene were described previously (Garraway et al., 2007). Candidate siRNAs were selected by the siRNA selection program from the Whitehead Institute for Biomedical Research (MIT) (Yuan et al., 2004). The sense and antisense sequence of the siRNA were joined by a spacer (TTCAAGAGA; SEQ ID NO:766) (Brummelkamp et al., 2002) to create a “stem-loop” sequence. Synthetic DNA oligomers with the corresponding sequences were ordered (Sigma-Genosys, St. Louis, Mo.), cloned into a serotype-2 recombinant adeno-associated virus (rAAV) plasmid and subject to the psiCHECK Dual Luciferase Reporter Assay (Promega, Madison, Wis.). Three plasmids expressing the most active siRNAs identified in the psiCHECK assay and one plasmid expressing a scrambled control siRNA were packaged into serotype-2 rAAV vectors (Musatov et al., 2002) and used in the in vivo studies.

The three active sequences (sense strand) identified by the psiCHECK assay were

ERK2-5, (SEQ ID NO: 765) 5′-GGAACAGGTTGTTCCCAAA, ERK2-7, (SEQ ID NO: 769) 5′-GGAGCAGTATTATGACCCA, and ERK2-8, (SEQ ID NO: 770) 5′-GACTGCTAGATTCCAGCCA, targeting regions spanning 1063-1081, 1178-1196 and 1289-1307 on the mouse ERK2 cDNA (GenBank accession number NM_011949) respectively. The sequence of this mouse ERK2 cDNA is reproduced below for easy reference (SEQ ID NO:771).

   1 GACGCGAACC CTTCCCTCCT CCCACTCGTA GCCCGCCCGT   41 CAGGCAGGAA GGCTGGCAGT GGTTCTACCG GCGGTTAATT   81 CTCTCCTCTG TGTTGTCCTC CTTCCTCGTT CCCGATCGCC  121 GGCGGGGGCG GCTACACGGG CGGCAGCGCG GTTCCTGCGG  161 GAAGCGCAGC ATAAGTCGAG CGGCAGCCGC GAAGCGTCGA  201 ACCGAACGCG GCGGCGGCGG CGGCGGCGGC GGCTGTGCAG  241 CCAACATGGC GGCGGCGGCG GCGGCGGGCC CGGAGATGGT  281 CCGCGGGCAG GTGTTCGACG TAGGGCCGCG CTACACCAAC  321 CTCTCGTACA TCGGAGAAGG CGCCTACGGC ATGGTTTGCT  361 CTGCTTATGA TAATCTCAAC AAAGTTCGAG TTGCTATCAA  401 GAAAATCAGT CCTTTTGAGC ACCAGACCTA CTGTCAAAGA  441 ACCCTAAGAG AGATAAAAAT CTTACTGCGC TTCAGACATG  481 AGAACATCAT TGGCATCAAT GACATCATCC GGGCACCAAC  521 CATTGAGCAA ATGAAAGATG TATATATAGT ACAGGACCTC  561 ATGGAGACGG ACCTTTACAA GCTCTTGAAG ACACAGCACC  601 TCAGCAATGA CCACATCTGC TATTTTCTTT ATCAGATCCT  641 GAGAGGGCTA AAGTATATCC ATTCAGCTAA CGTTCTGCAC  681 CGTGACCTCA AGCCTTCCAA CCTCCTGCTG AACACCACTT  721 GTGATCTCAA GATCTGTGAC TTTGGCCTTG CCCGTGTTGC  761 AGATCCAGAT CATGATCACA CAGGGTTCTT GACAGAGTAC  801 GTAGCCACAC GTTGGTACAG AGCTCCAGAA ATTATGTTGA  841 ATTCCAAGGG TTATACCAAG TCCATTGATA TTTGGTCTGT  881 GGGCTGCATC CTGGCAGAGA TGCTATCCAA CAGGCCTATC  921 TTCCCAGGAA AGCATTACCT TGACCAGCTG AATCACATCC  961 TGGGTATTCT TGGATCTCCA TCACAGGAAG ATCTGAATTG 1001 TATAATAAAT TTAAAAGCTA GAAACTATTT GCTTTCTCTC 1041 CCGCACAAAA ATAAGGTGCC ATGGAACAGG TTGTTCCCAA 1081 ATGCTGACTC CAAAGCTCTG GATTTACTGG ATAAAATGTT 1121 GACATTTAAC CCTCACAAGA GGATTGAAGT TGAACAGGCT 1161 CTGGCCCACC CATACCTGGA GCAGTATTAT GACCCAAGTG 1201 ATGAGCCCAT TGCTGAAGCG CCATTCAAGT TTGACATGGA 1241 GTTGGACGAC TTACCTAAGG AGAAGCTCAA AGAACTCATT 1281 TTTGAAGAGA CTGCTAGATT CCAGCCAGGA TACAGATCTT 1321 AAATTGGTCA GGACAAGGGC TCAGAGGACT GGACGAGTTC 1361 AGATGTCGGT GTCCCCCCAG TTCTTTACCC TGGTCCTGTC 1401 TTCAGCCCGT CTCAGCTTAC CCACTCTTGA CTCCTTTGAG 1441 CCTTTCAGAG GGGCAGTTTC TGGTAGTAGC AGCTTTTATA 1481 CTTTCACGGA ATTCCTTCAG TCCAGAGAGT TCTGGCAGCA 1521 GGCCGTGCAG CAGTGTGCAC TTTCAATGCA CTTAACTGCT 1561 TACTGTTGTT TAGTCACGAA CTGCTTTCTG GTTTGAAAGA 1601 TGCAGTGGTT CCTCCCTGTT CTGAATCCTT TCTCCATATC 1641 ATGTGCTGAA CCATCAGCCT CATCAGAGGG AGAGTCTTTC 1681 CAGACTTGTT CCAGTTACTG GCACCTCACT TCACAGTAGG 1721 GAGGCTAGGA CATAAGGCAC CTTAAGTCAG TGACAGCTCC 1761 AAATTTGCAC TTCATCTGTT GGCTAGTAAC TGTCTACCTA 1801 GACAGTAGGA GCTTGTGGGT ATCCCTGGAT GGTATTACAG 1841 GCTACAGGGG CAGGGGCTTC TGTTGCAGGA GTCCTTTGGG 1881 GCTATTTTCC TGTGTATCAT GTTAGTCCTA AATTTAAGGT 1921 ATGTACTATT TGCCCAGCTT TTTAAAAATT TGATCATTGT 1961 TTAAATGAAA TAGGAAGGAA GCATTGCACC AGCAGTATCT 2001 GTTGTTCTGC AGATTTTATA TGGTTACTTG TATCGTAATG 2041 GAGGTGGAGC TCTTGCCAAA ATGTTACATG CTATCCTTAG 2081 CCAGAGAGTG AAAGTAACAG CTGTGCTTGT CATTTACTGA 2121 AAGGTGGACA CACACAAAGC TGTGGAAGTT TCCAGAACAG 2161 TAGAGAGCAA GCTGACCTAG ATGTTCAGGG CAGAGCTCCA 2201 TATAACCTTG AACAGCCACA CAGAAGGCTG TTTGCGTAAC 2241 CACATTCACT ACCTAGGGAT TTAGCTAAAA GGAACACTGC 2281 ATCTTTAAAT GAGAAAGTGT ACAGTTCTTC TCCTGCAGCA 2321 TGTCAGCATC TCGAGCTCAC TTTTCAGCAG TGTAATGACT 2361 TGTATGTAAT AAAGCCTTGA TGGGCTCTCC TCATGAAGCT 2401 CTGCTCTGTT GCCAAGTTAG AGATGTTTCT GGTACTGCTG 2441 AGTTAATGTC ATAAAAGGCT AGCAGTAACT GTTCGAGCTC 2481 TCTTTTATTT CCTTCTCTCC TATATTTTGT TCCTGCACTG 2521 TGTGCTGTGG AGTTGATGGT GTTATCCCAG TGCGGTGCCT 2561 CCAGACCCCC TCACTGCTCT CTGATGAGAA ATATGCCTTG 2601 TTCAATACTT ACTGTGCTCT TGCATGACTG TTAAGGTTTC 2641 TGTGCAGAGA CCAATGTCCA AGTGTCACAT CCTTTGATTG 2681 AACGAAATCT GTTGTGACCT CTGAGTTGTA TTCCATGAAG 2721 AGAATGCTAC CCAGAAGATA ATGTAGAAAA GATAATTATA 2761 TTGTTAACTT TTCATTTCTC AGCTGTCCTT TTGTTTTCTT 2801 GGTTTTTATT TTTTATTTTG ACATCAATGG AAAATGGGTT 2841 CTATAAAGAC TGCCTGCTAG TATGAACAGC AATGCAATGC 2881 ACTTGTAACT CATGGAAATA AATGTACATC TTTATCTTTA 2921 CACCCATGAT AAGATTCAGT GTTGATTTTC TCTGGATTGG 2961 TGTGTCCTAA GTAGGCACTC ATAATCAATT TATGGCTTGT 3001 GCTTCAGACA AAAATGTTCA TGGGCCTTAC TCTACTTCTC 3041 CCCACTCCAC CCTACCCCCC ATGCACTGCC CCTCACAGCA 3081 GTTTACGTAT ATGGCTGGGA AAGGTCCTTT TCAGCTGCAC 3121 ATGGTGCCAT GCATCGTTAA TCCCAGCATT CAGAAGTCAG 3161 AGGCAGGTGG ATCTCTGAAT GGAAGCAGGC CTGATTTGCA 3201 TAGGGAGGTC CAAGACAGCT GGAACTCTAT AGGTCCTGTC 3241 TCAAAAAAAA CAGAGTCCTC CCCGTCTGCC TCTCAGCAGC 3281 AAATGAATCT GACATGATCC TCTCTAAAAC AGGTCTCAAG 3321 TAGTCAGATG TTGATGATGG CACCCAAACA TGCCCAAGTT 3361 AGGATCTGGT TCCCTCTGAA AAGGGCCTTC TTGCCTCTGT 3401 ATCCTAGAGC TGTAGGAAGG GCTGTTCAAG ATCTCATGTA 3441 CCTGCTACCA AGTTCAAGGT AGCACATACC TCACCTGGCT 3481 AAAGAATGGC TGACTCATCC CAGAAACCAG ATCTCAGTTC 3521 TTGGCCTAAA TCCCTGCTTT TCACTTCCAC ACATGAAGCC 3561 CACTGGCATT GAAGGAATAG AGGTTCAGCT TTCATTGATA 3601 CAGTAGTGGT CAGTTTTCCT TTTTCTTTTT GTCTTTTTTT 3641 TTAAAGCACT GACTGTTCTC CTACTTGTTT CTTTTTCATA 3681 TTTTTAATCC CATGAGATTA ATTTGCATTC TTGTGAATAA 3721 GGAAACCATA GCCTCATCTT CTCGAGGTCT GAGCTTTCTG 3761 CCCTTCCTGG CACTGTGGAG AGGGGTTGGT GTGAGATCAC 3801 TCACTTCATC CTAGTCACTG TATCACAAGT GTGGCTTTCA 3841 TGTAGCCATT GTAAATGACA GCTCAGAGCT GTCAGGTATA 3881 GAAACGCTCA TTATTTTGGT TCTCATGTTT CTAAAAATGT 3921 TTGGATAACG TCATCTGCAT ACTGGTGTCA TTGGGTGCCT 3961 CTACTATTCA TACACATAGA TAAGCTGTCT GGTGGATGGG 4001 CTTTTTGTCC AAGTCTTAAT ATGTGAGGGA AAAAAACCCA 4041 AAAACATGAA AACATTTAGC ATGAAGAAGA TAGCTATCCA 4081 ACAATCCCAG AGCGCTTGAT GATACCGGCA TTCAGAGCTG 4121 ACACTGACCT ACTCTGTGGT GCATTTATTC TGCCCCCACC 4161 CTCATCCCTC TCATTTGAGG ACAGGCAACA CTTGGGCTGG 4201 GCATGACTGT TAGTTTTGGG AAGCTGTGAA TTAACAGCAG 4241 CTATCTCTGA GGAATCACAA AGGTAGACAC CTACACTGCA 4281 TGCCACATAG TATTCAGACC ACTTAGGGAG ACTTCCATTT 4321 GCTTAGGATA ATATTTACAT TAATATTAGT AGTTAGGTTT 4361 GAACTTTTGG TGACTTCTAT ACTACGGTAA CACATTCATA 4401 TATGCATATG CTTTGGGTCC TTCATACTAC TTTTATATTT 4441 GTAAATCAGT GTTTTGGAGC AATTCCAAGT TTAAGGGAAA 4481 TATTTTTGTA AATGTGATGG TTTTGAAAAT CTGAGCAATT 4521 CTTTTGCTTA CAAGTTTTTT TAAAGCATTT GTGCTTTAAA 4561 ATTGTGCTAG TGTTTGGAAT ATGATACCCT ATAACCCAGA 4601 TAAGAAACAT AAGAATGGAG TAAACGCTGT CGCTTGTCGT 4641 GCTATGCCCA GCTTGGCGTG CTGGATCAGC AGTGGGACTC 4681 CGGAGTCCCT AGGGTCACAC CAGCTCACCT GCAGCTTGTT 4721 GCCTTTCTGT GCCGTCCGCC CGCCCTTCAG AGCACTCCAG 4781 AAAGTTCTGA CATGGCTCTG TATCTGCTCT GTACTGTGGA 4801 TGCCTTTTTG GTGTTGTATC CCAAACTGCA TAGATTATTT 4841 AGGATAATGA TAAGTTTAAA AAATTAATGT TGAAGAAAGA 4881 TTTTATTAAG AATTTAAATG TTTTTTCATT ATATTGTTAA 4921 ACTTGAACAT TTATCTGTGG CTTATGTGAT TTGGTTAATA 4961 TGTATAAAAA TTGTAAGAGG TTTATATTTC ATCTTAATTC 5001 TTTTGATGTT GTAAACGTGC TTTTCAATTC ATTATTTGAA 5041 TGTTTATGGC ACCTGACTTG TAAAAAGAAT TACAAAAAAA 5081 AAAATCCTTA GAATCATTA A scrambled ERK2-7 sequence (MM), 5′-ACCCAGTATTATGACGAGG (SEQ ID NO:772) was used as the control.

In Vivo Delivery of the rAAV Vectors.

The viral vectors, ERK2-5, 2-7, 2-8 and MM were micro-injected into the spinal cord dorsal horn (SCDH) as described by South et al. (South et al., 2003). The mice were anesthetized with ketamine/xylazine. A laminectomy was performed to remove part of the dorsal L2 and L3 spinous process and the lumbar area of spinal cord was exposed for intraparenchymal injection (IPI). Three unilateral injections of 1 μl (1-3×10⁹ viral particles/μl) were administered 0.5-0.7 mm apart, at a depth of 0.3 mm from the dorsal border and 0.5 mm from the midline, using a glass pipette with a 40-μm-diameter tip attached to a 5 μl Hamilton syringe. The syringe was mounted on a microinjector (David Kopf Instruments, Tujunga, Calif.) attached to a stereotaxic unit (David Kopf Instruments). After the injection, the overlying muscles were closed with 5-0 chromic gut and the skins were closed with wound clips. Animals were allowed to recover for three weeks before undergoing behavioral tests, or sacrificed for histological and Western blot analysis.

Immunohistochemistry (IHC).

Mice were anesthetized with pentobarbital and then perfused transcardially with 4% paraformaldehyde (PFA) in phosphate buffered saline (PBS) with 1 mM NaF. Fifty mL of fixative was perfused over 5 min by using a peristaltic pump. The spinal cord was dissected and placed in 4% PFA for 1 hr before being transferred to 30% sucrose for cryoprotection for 72 hr. Lumbar spinal cord cryosections of 20-μm thick were obtained from a cryostat (Leica, Bannockburn, Ill.) for IHC and in situ hybridization.

For IHC, the spinal cord sections were incubated in blocking solution (3% normal goat serum, 0.1% Triton X-100 in Tris buffered saline) for 30 min. After washing in Tris buffered saline (TBS), sections were incubated with one or two of the following primary antibodies: rabbit anti-GFP (1:1000; Invitrogen-Molecular Probes Inc, Eugene, Oreg.), rabbit anti-phospho-ERK1/2 (1:1000; Cell Signaling Technology, Inc., Danvers, Mass.), mouse anti-NeuN (1:400; Millipore-Chemicon, Bedford, Mass.), rabbit anti-c-fos (1:2000, Santa Cruz Biotechnology, Santa Cruz, Calif.), rabbit anti-Dynorphin A(1:2000, Bachem, San Carlos, Calif.), mouse anti-GFAP (1:2000; Millipore-Chemicon) and rat anti-OX42 (1:1000, BD Biosciences, San Diego, Calif.) overnight in blocking solution at 4° C. The sections were washed in TBS and then incubated in appropriate fluorescent secondary antibodies or biotinylated goat anti-rabbit or anti-mouse IgG (1:250; Vector Laboratories, Burlingame, Calif.) in blocking solution. Biotin slides were further incubated with 3,3-diaminobenzidinetetra-hydrochloride (DAB). Fluorescent slides were mounted in the anti-fading mounting medium GelMount (Invitrogen, Eugene, Oreg.). DAB slides were dehydrated through a series of ethanol and xylenes, then coverslipped in Permount (Thermo Fisher Scientific, Inc., Waltham, Mass.). To minimize variability in staining, tissues from all treatment groups were run in the same session. A negative control was performed using diluted normal goat serum instead of the primary antibody.

Non-Radioactive In Situ Hybridization.

ERK2 mRNA expression in the SCDH was detected by non-radioactive in situ hybridization as described by (Garraway et al., 2007). Digoxigenin (DIG) labeled antisense or sense riboprobes were synthesized using an in vitro transcription kit (Roche Applied Science, Indianapolis, Ind.) from the mouse ERK2 cDNA (kindly provided by Dr. Michael J. Weber, University of Virginia). On day 1, slide mounted cryosections of spinal cord (20 μm) were incubated in the following: (1) 4% paraformaldehyde (PFA) in phosphate buffer saline (PBS) for 20 min; (2) PBS, three times for 5 min each; (3) Proteinase K solution (Sigma) for 2 min; (4) PBS, twice for 5 min each; (5) 4% PFA in PBS for 5 min; (6) PBS for 5 min; (7) 0.25% acetic anhydride in 0.1 M Triethanolamine for 10 min; (8) 2×SSC, twice for 5 min each. Next sections were incubated in pre-hybridization solution (50% formamide, 0.3M NaCl, 10 mM Tris pH 8.0, 1 mM EDTA, pH 8.0, 500 μg/ml salmon sperm DNA and 500 μg/ml yeast tRNA) at 55° C. in a chamber containing towels moistened with 4×SSC and 50% formamide. After incubation for 2 hr, the pre-hybridization solution was drained and sections were hybridized with DIG-labeled antisense or sense probes for ERK2 (1:1000), coverslipped, and placed in a 55° C. oven overnight. On day 2, the coverslips were removed and the hybridized spinal cord sections were sequentially incubated in the following: (1) 5×SSC at 55° C. for 10 min; (2) 50% formamide in 2×SSC at 55° C. for 20 min; (3) RNAse buffer at 37° C., twice for 5 min each; (4) RNAse A (50 μg/mL, Sigma) at 37° C. for 30 min; (5) RNAse buffer at 37° C. for 15 min; (6) 50% formamide, 2×SSC at 55° C. for 20 min; (7) 2×SSC, twice for 15 min each; (8) washing buffer for 10 min; (9) blocking solution for 30 min; (10) anti-DIG antisera conjugated to alkaline phosphatase (1:500) for 2 hr; (11) washing buffer, twice for 15 min each; (12) detection buffer for 5 min; (13) nitro blue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) overnight. On day 3, slides were rinsed in distilled water and dehydrated through a graded ethanol series, xylenes, and coverslipped in Permount (Thermo Fisher Scientific, Inc.).

Microscopic Analysis.

Fluorescent IHC images were captured by a Zeiss LSM 510 laser scanning confocal microscope. Bright field IHC and in situ images were captured by a Nikon Eclipse 80i microscope (Nikon, Melville, N.Y.) equipped with a digital CoolSnap camera (Photometrics, Huntington Beach, Calif.) through an interface between the camera and a Macintosh computer using the IPlab software (BD Biosciences Bioimaging, Rockville, Md.). Quantitative analysis was performed by a blinded observer using Metamorph software (Universal Imaging, Downingtown, Pa.) as described previously (Garraway et al., 2007). A total of 4-5 sections spacing 400-500 μm apart were used for each animal. At least three animals were included in each treatment group.

Immunoblot.

Animals were deeply anesthetized by isoflurane, decapitated and the lumbar spinal cord dorsal horn at the level of L4-L6 were rapidly dissected. The right and left dorsal horn were separated and immediately homogenized in modified RIPA buffer (50 mM Tris-HCl, pH7.4, 1% NP40, 1 mM EDTA, 150 mM NaCl) supplemented with protease inhibitor cocktail (Sigma) diluted 1:10, 2 mM PMSF, 2 mM NaF and phosphatase inhibitor cocktail I and II (Sigma), then frozen in liquid nitrogen. After all the samples were collected, tissues were thawed on ice, sonicated and centrifuged at 4° C. at 14000 g for 10 min to obtain the supernatant. The protein level of each sample was measured by the BioRad DC assay. Samples were then diluted in Laemmli sample buffer on the same day to a final concentration of 2 ug/ul, boiled for 5 min and stored at −80° C.

The spinal cord dorsal horn samples were separated on SDS-PAGE gels (10% Tris-HCl gel; Bio-Rad, Hercules, Calif.) and transferred to polyvinylidene difluoride filters (PVDF, Millipore, Bedford, Mass.), which were subsequently blocked in blocking solution (5% dry milk in TBS with 0.1% Tween-20) for at least an hour. Membranes were incubated with rabbit anti-pERK1/2 antibody (1:1000; 07-362; Cell Signaling Technology, Inc.) in blocking solution overnight at 4° C., washed in TBS with 0.1% Tween-20 (TBST), then incubated with HRP-conjugated anti-rabbit IgG (1:1000, Pierce Biotechnology, Inc., Rockford, Ill.) in blocking solution for 1 hr. Membranes were washed with TBST followed by TBS and developed using SuperSignal West Pico ECL kit (Pierce Biotechnology, Inc.), then exposed to film (Kodak, Rochester, N.Y.) for various times. Next, membranes were stripped (Pierce) and reprobed for ERK1/2 using rabbit anti-ERK1/2 antibody (1:5000, Cell Signaling Technology, Inc.) followed by HRP-conjugated anti-rabbit secondary antibody (1:10,000, Pierce Biotechnology, Inc.). For loading control, blots were further stripped and re-probed for β-actin using a mouse monoclonal antibody (1:50,000, Sigma) followed by HRP-conjugated anti-mouse secondary antibody (1:200,000, Pierce Biotechnology, Inc.). Exposures yielding signal intensity in the linear range without saturation were used for densitometry analysis with Fluorchem 9900 (Alpha Innotech, San Leandro, Calif.). Ratios of intensity of pERK1/2 or ERK1/2 to β-actin were calculated, normalized to the control samples and subject to statistical analysis. At least 4 animals were included in each treatment group.

Behavioral Testing.

All behavioral testing was conducted in groups of 10 mice per treatment by a blinded observer. Motor function tests were performed before mechanical stimulus threshold or thermal paw withdrawal threshold were measured (Garraway et al., 2007). No deficits in reflexes for surface righting, placing/stepping and grasping/climbing were found in animals treated with either the control rAAV vector or rAAV viral vectors.

Mechanical stimulus threshold to a non-noxious mechanical stimulus was determined by paw withdrawal to the application of a series of calibrated von Frey filaments to the surface of the hind paws. The animals were placed in a Plexiglas cage with mesh flooring suspended above the researcher and left to acclimate for 30 min. von Frey filaments were applied perpendicularly against the mid-plantar surface of the foot. The “up-down” method of Dixon (Chaplan et al., 1994) was used to determine the value at which paw withdrawal occurred 50% of the time, interpreted as the mechanical threshold.

Thermal paw withdrawal threshold was assessed using a thermal nociceptive stimulus apparatus (Hargreaves et al., 1988). Prior to the test, the animals were allowed to acclimate for 30 min to the test chamber on a pre-heated glass plate maintained at 30° C. A radiant thermal stimulus (5.10 amps) was applied to the mid-plantar surface of the hind paw through the glass plate. The latency, in seconds, for the withdrawal of the paw from the heat source was determined automatically. If no response was elicited, the heat source was automatically shut off at 20 sec to prevent tissue injury. A minimum break of 5 min was allowed between each trial. Three to six trials were performed for each paw.

Fifteen μl of Complete Freund's adjuvant (CFA, Sigma) was injected into the right hind paws of lightly restrained mice. The mice had received either the control vector MM or siRNA vector 2-7 in the right spinal cord dorsal horn at least three weeks prior to the intraplantar injection of CFA. Mechanical stimulus threshold, thermal paw withdrawal threshold and paw size were measured before (baseline) and at 24, 48 and 96 hr after CFA.

Statistical Analysis.

The immunohistochemical, in situ hybridization, Western blot and behavioral data were analyzed by one-way ANOVA followed by the Student-Newman-Keuls test (multiple groups) or the t test (two groups) using the InStat software (GraphPad, version 3.00, San Diego, Calif.). The data are represented as mean±SEM.

Example 2 Reduction of ERK2 Expression Inhibits Pain in Mice

This Example describes the effects of inhibiting ERK2 expression by siRNAs that are specifically targeted to ERK2.

Knockdown of ERK2 Expression in the Lumbar Spinal Cord Dorsal Horn Neurons by rAAV Vectors Expressing Active siRNAs.

Three serotype-2 rAAV vectors (vector 2-5, 2-7 and 2-8) expressing ERK2 siRNAs and one control vector expressing a scrambled siRNA (vector MM) were used in the current study. The psiCHECK Dual Luciferase Assay was performed before the virus preparation and injection to examine the ability of vectors to inhibit ERK2 expression in cultured cells. Compared to a control rAAV plasmid that did not express an siRNA, all three active siRNA vector plasmids significantly inhibited the activity of Renilla luciferase, which was translated from a fusion mRNA containing the ERK2 and luciferase cDNA sequences. The control MM vector was ineffective in this assay (FIG. 1J).

The rAAV vectors were injected intraparenchymally into the spinal cord dorsal horn of adult mice. Three weeks after intraparenchymal injection of rAAV vectors, a robust and spatially localized expression of GFP was observed on the ipsilateral side of lumbar spinal cord dorsal horn (FIG. 1A-D). The expression of GFP resulting from each of the four rAAV vectors extended for more than 3 mm rostrocaudally, encompassing the full L4, L5 and L6 spinal segments in mouse. In situ hybridization was performed on slides adjacent to the GFP slides to examine the mRNA expression of ERK2. On the side contralateral to the vector injection, ERK2 mRNA was observed in the gray matter. Vector MM did not alter the ERK2 mRNA level (FIG. 1E) in the ipsilateral dorsal horn. In contrast, there was a nearly complete depletion of ERK2 mRNA in the region corresponding to the GFP immunolabeling in animals treated with vector 2-5, 2-7 or 2-8 (FIGS. 1F-H). A densitometry analysis of the in situ images showed that the extent of ERK2 mRNA knockdown was similar among the three active siRNA vectors, ranging from 75% to 80% reduction on the ipsilateral side compared to the contralateral side (II, p<0.05, vs. vector MM).

To determine whether vector administration resulted in glial activation, sections of SCDH were compared from mice that were untreated or had received either the vector MM or the active siRNA vector 2-7 at 3 weeks before the analysis. One example of the immunolabeling is shown in FIGS. 2G1-2G9. Compared with sections from untreated mice, there was no evidence of hypertrophy, thickened processes, or enlarged cell bodies in GFAP-labeled astrocytes (Guo et al., 2007) or the shortened, thickened processes seen with activated microglia labeled with OX42 (Raghavendra et al., 2004). Examination of sections at a higher magnification than is shown in FIGS. 2G1-2G9 supported these observations (data not shown). There was no evidence of neuronal cell loss, a finding consistent with a previous observation that vector derived siRNAs did not result in loss of nuclei in the spinal cord dorsal horn (Garraway et al., 2007).

GFP immunolabeling was colocalized with NeuN, a neuronal marker, but not with GFAP, an astroglial marker, or with OX42, a microglial marker, as revealed by confocal fluorescent immunohistochemistry in animals that received the control MM vector (FIG. 3G1-G9) or siRNA vector 2-7 (FIG. 3H1-H9). There was no evidence of gliosis in the spinal cord dorsal horn of animals treated with either vector.

Due to the high sequence similarity between the ERK1 and ERK2 proteins and the lack of a specific ERK2 antiserum, immunohistochemistry was not able to distinguish between ERK1 and ERK 2 expression. Therefore, Western blot analysis was used to quantify the knockdown of ERK2 expression. Compared to the control vector MM, vector 2-7 induced greater than 50% reduction in the expression of ERK2 in the ipsilateral spinal cord dorsal horn (FIGS. 2A and B, p<0.05), while the expression of ERK1 was not affected (FIGS. 2A and C). This knockdown was not present on the contralateral side (FIGS. 2A and B). As expected, the level of phospho-ERK2 (pERK2), the active form of ERK2, was also decreased on the ipsilateral side in animals treated with vector 2-7 compared to control vector MM (FIGS. 2D and 2E, p<0.05). Interestingly, there was an accompanying increase in pERK1 in blots from these animals (FIGS. 2D and 2F, p<0.05), suggesting a loss of competition between ERK1 and ERK2 for their binding and activation by MEK. Vector MM did not have any effect on the expression or phosphorylation of ERK1 and ERK2 compared to animals that did not receive intraparenchymal injection of a vector (FIG. 5A-E).

The cellular localization of pERK1/2 on spinal sections was examined using an antibody that recognizes both pERK1 and pERK2. In animals that received the control vector MM, pERK1/2 was observed in the lumbar spinal cord dorsal horn, mainly in lamina I and II (FIG. 3A). Confocal double fluorescent immunohistochemistry revealed that pERK1/2 immunolabeling was co-localized with NeuN (FIG. 3A-C) but not with GFAP or OX42 (data not shown). In animals treated with siRNA vector 2-7, the pERK1/2 immunolabeling was dramatically decreased in the ipsilateral dorsal horn (FIG. 3D-F), suggesting that pERK1/2 immunolabeling is mainly comprised of pERK2. As stated previously, an increase in pERK1 accompanying ERK2 knockdown was observed. However, the increase in phosphorylation of ERK1 was apparently not sufficient to overcome the knockdown of ERK2 expression and phosphorylation in these spinal cord dorsal horn neurons.

Knockdown of ERK2 Expression Prevented ERK Activation in the Spinal Cord Dorsal Horn Following Intraplantar CFA Administration.

Injection of the Complete Freund's Adjuvant (CFA) into the hindpaw of mouse induces a rapid increase in the phosphorylation of ERK1 and ERK2 in the ipsilateral lumbar spinal cord dorsal horn, which is then maintained for at least 7 days (Ji et al., 2002; Adwanikar et al., 2004). This induction is associated with the development of hyperalgesia and allodynia in the injected paw. ERK1/2 may regulate their targets by either post-translational or transcriptional mechanisms, presumably at different stages of the injury-induced pain. Therefore, ERK1/2 phosphorylation was examined in the lumbar spinal cord dorsal horn at both 1 hour and 96 hour after injection of the Complete Freund's Adjuvant (CFA).

As illustrated in FIG. 4A, Western blot analysis showed a significant reduction in ERK2 in the ipsilateral spinal cord dorsal horn after administration of vector 2-7 compared to the control MM vector. The reduction was observed at 1 hr and maintained to 96 hr after CFA injection (p<0.05, vs. vector MM). In the control animals, ERK2 was increased at 96 hr after CFA in the ipsilateral spinal cord dorsal horn compared to no treatment (NT) or 1 hr (FIGS. 4A and B, p<0.05). There was also an increase in ERK1 expression at 96 hr in the control animals compared to no treatment and 1 hr (FIGS. 4A and C, p<0.05). Vector 2-7 did not alter ERK1 expression and prevented the ERK1 increase induced by CFA (FIGS. 4A and C). In control mice (no vector treatment), CFA induced an increase in pERK2 at 1 h that persisted at 24 and 96 h after CFA (FIG. 5F).

There was a decrease in basal pERK2 after vector 2-7 was administered and this reduction persisted from 1 hr to 96 hr after CFA (FIGS. 4D and E, p<0.05, vs. vector MM). In contrast, pERK2 was increased at 1 hr and 96 hr after CFA in the control animals treated with vector MM (FIGS. 4D and E, p<0.05, vs. vector MM/NT). Basal pERK1 was increased as a result of intraparenchymal injection of vector 2-7. This increase was not altered by CFA at either 1 hr or 96 hr. In contrast, pERK1 was increased by CFA in the control vector MM group at 1 hr and 96 hr (FIGS. 4D and 4F, p<0.05, vs. vector MM/no treatment). These results clearly demonstrated that the increases in ERK2, ERK1 and pERK2 following CFA were prevented by vector 2-7, although vector 2-7 induced an increase in basal pERK1 level.

Next, 3,3-diaminobenzidinetetra-hydrochloride (DAB) immunohistochemistry was performed to quantify and localize pERK1/2 changes in the spinal cord dorsal horn after CFA. In the control vector MM group, immunolabeling of pERK1/2 was observed mainly in laminas I and II in the ipsilateral spinal cord dorsal horn. Intraplantar CFA injection induced a significant increase in the pERK1/2 labeling at 1 hr and 96 hr, measured as the number of pERK of labeled neurons in laminas I and II or percentage of field (FIGS. 6A-C,G,H) (p_0.05 vs vector MM/no treatment). The level of pERK1/2 immunolabeling in the vector 2-7 group was significantly lower than the control group at each corresponding time point (FIGS. 6D-G,H)(p<0.05 vs vector MM).

Effects of the Knockdown of Neuronal ERK2 in the Spinal Cord Dorsal Horn on Motor Reflexes, Acute Thermal and Mechanical Thresholds.

To investigate the functional consequences of the ERK2 knockdown, the motor reflexes, hind paw thermal withdrawal latency, and mechanical withdrawal threshold in animals were first examined before and after intraparenchymal injection of each viral vector. None of the animals that received either control vector MM or vector 2-7 exhibited any signs of motor deficits (data not shown). No change was observed in heat withdrawal latency or mechanical withdrawal threshold, when tested at least 3 weeks after the vector administration (FIGS. 7A and 7B), indicating that neither the intraparenchymal injection procedure nor knockdown of neuronal ERK2 in the spinal cord dorsal horn affected acute pain thresholds.

Knockdown of ERK2 in the Spinal Cord Dorsal Horn Prevented CFA-Induced Pain.

After intraplantar CFA administration, there was an equal increase in paw size in animals treated with either the control vector MM or the siRNA vector 2-7 (FIG. 8A, p<0.05, vs. baseline, n=10 per treatment group), indicating a similar degree of peripheral inflammation. However, only the vector control animals exhibited significant decreases in the thermal withdrawal latency (thermal hyperalgesia) (FIG. 8B, p<0.05) and mechanical withdrawal threshold (mechanical allodynia) (FIG. 8C, p<0.05) at 24, 48 and 96 hr after CFA. The ERK2 knockdown in the spinal cord dorsal horn protected the animals from developing thermal hyperalgesia and mechanical allodynia for up to 96 hr after CFA (FIGS. 8B and 8C).

The Expression of c-Fos and Dynorphin A Following CFA.

To investigate how ERK2 knockdown may prevent CFA-induced inflammatory pain, the expression of c-fos (FIGS. 9A-9G) and dynorphin A (FIG. 10) was examined. These two genes may be regulated by ERK via CRE-mediated mechanism and lead to long-lasting synaptic modifications in pain (Ji et al., 2002; Obata et al., 2003). A very low level of c-fos immunolabeling was observed before CFA. At 1 hour after CFA, the expression of c-fos was strongly induced in the ipsilateral spinal cord dorsal horn. This induction was equal in both groups of animals and diminished at 96 hr. Dynorphin A immunolabeling was not different in animals that received vector 2-7 compared with the control animals. In addition, no changes were observed in the level of dynorphin A at either 1 hr or 96 hr after CFA. These results indicate that the expression of c-fos or dynorphin A is unlikely to be regulated by neuronal ERK2 at either 1 hr or 96 hr following CFA.

This is the first report of spatial-temporal knockdown of ERK2 gene expression mediated by a siRNA in the spinal cord dorsal horn of adult mice. The ERK2 siRNAs delivered by a neurotropic rAAV vector produced a localized reduction in the basal level of both ERK2 and its phosphorylated form (pERK2) in spinal cord dorsal horn neurons. The increase in both ERK2 and pERK2 induced by intraplantar CFA in the spinal cord dorsal horn of control mice was prevented by the ERK2 siRNA. In addition, the ERK2 siRNA vector protected the mice from CFA-induced thermal hyperalgesia and mechanical allodynia for at least 96 h.

The rAAV vector-based siRNA approach presents a potent and facile tool to produce a spatial and temporal knockdown of the expression of a gene of interest (Garraway et al., 2007). Several factors dictated the choice of a rAAV vector for the delivery of the ERK2 siRNA. First, the serotype-2 rAAV vector used in the current study selectively transduces neurons in vivo (Kaspar et al., 2002). Second, rAAV is able to mediate long-term siRNA expression and gene knockdown in the transduced cells. Although the GFP and ERK expression was examined for only 6 weeks, previous studies by the inventors demonstrated that a single administration of a rAAV vector resulted in the knockdown of NR1 gene expression that persisted for at least 6 months (Garraway et al., 2007). Third, rAAV is safe and therefore convenient to use in behavioral experiments requiring repeated measurements. Fourth, rAAVmediated gene knockdown could be controlled both temporally and spatially. This conditional approach avoids embryonic lethality associated with a constitutive knock-out of ERK2 (Hatano et al., 2003; Saba-El-Leil et al., 2003; Yao et al., 2003).

Consistent with the observations described herein, several reports (Kaspar et al., 2002; South et al., 2003; Garraway et al., 2007) have provided evidence at the ultrastructural and light microscope levels as well as direct behavioral threshold evidence that the injection of AAV into the brain or spinal cord dorsal horn does not result in significant immune or glial activation or behavioral sensitization. It has been reported that a high dose of a siRNA might induce nonspecific and off-target effects (Bridge et al., 2003; Sledz et al., 2003). However, previous studies by the inventors indicated that neither an NR1 siRNA nor a control siRNA delivered by the rAAV vector induced detectable cellular toxicity (Garraway et al., 2007). Transduced neurons exhibited unaltered expression of NeuN compared with the contralateral side. In addition, no any signs of gliosis or neuronal damage were observed (FIG. 2G). The three vectors expressing different siRNAs induced a similar degree of GFP expression and knockdown of the ERK2 mRNA in the spinal cord dorsal horn, suggesting the knockdown is unlikely to be induced by nonspecific or off-target effects. The knockdown clearly targets the ERK2mRNA and protein as revealed by in situ hybridization and Western blot. The closely related ERK1 was unaffected at the protein level.

Thus, the data clearly show that the ERK2 siRNA vector 2-7 greatly reduced basal and induced pERK immunolabeling in the spinal cord dorsal horn (FIG. 3A,D) and that this pERK species was pERK2 as measured by Western blot (FIG. 2D). Moreover, the ERK2 knockdown completely blocked CFA-induced thermal hyperalgesia and mechanical allodynia. Additional experiments examining inflammatory pain behaviors earlier than 24 hour after CFA are required to demonstrate the role of ERK2 in the early induction of inflammatory pain.

Injury-inducing stimuli such as intraplantar CFA injection result in a rapid activation of ERK1/2 in the spinal cord dorsal horn. NMDA receptors play a major role in ERK1/2 activation (Ji et al., 1999; Cheng et al., 2008), although other players are also involved (Kawasaki et al., 2004). This activation of ERK1/2 after CFA is sustained at 24 h and persists for at least 96 h. Several sources may contribute to the prolonged ERK1/2 activation, such as sustained primary afferent input from the periphery. The injected hindpaw remains swollen at 96 h, indicating an ongoing peripheral inflammation. Another source could be the descending excitatory pathway from supraspinal sites (Svensson et al., 2006).

ERK1/2 can phosphorylate several pain-related proteins including the NR1 subunit of the NMDA receptor (Krapivinsky et al., 2003) and the Kv4.2 potassium channel (Hu et al., 2006). Phosphorylation of these proteins can contribute to the central sensitization in spinal neurons after peripheral injury, which leads to increased membrane excitability in the affected neurons. In addition to its role in the posttranslational regulation, ERK1/2 may also maintain pain hypersensitivity by promoting transcription of genes that are important for neuronal plasticity. A major transcription factor activated by ERK1/2 is cAMP response element-binding protein (CREB), which in turn induces transcriptional activation of many genes such as c-fos, TrkB (Obata et al., 2003), NK-1, and prodynorphin (Ji et al., 2002) via CRE-mediated mechanism. As described above, the immediate-early gene c-fos was activated in the SCDH in control animals at 1 h but not 96 h after CFA. However, a comparable change was observed in the ERK2 knockdown animals, indicating c-fos was activated by ERK2-independent mechanisms. No changes were detected in the level of dynorphin A at 1 or 96 h after CFA.

ERK1 and ERK2 mRNA levels are upregulated at 12 h after formalin (Li et al., 2004). We found the expression of ERK1 and ERK2 protein remains upregulated at 96 h after intraplantar CFA in control animals, but was prevented by the ERK2 siRNA vector.

Example 3 Reduction of ERK2 Expression in Human Cells

This Example illustrates that ERK2 expression can be inhibited by siRNAs that are specifically targeted to human ERK2.

Separate cultures of an immortalized cell line of human embryonic kidney cells (HEK293) were transfected with three active siRNAs directed against human ERK2. Another culture of HEK293 cells was transfected with an inactive control mismatch (MM) siRNA. The sequences of the three human ERK2 siRNAs (#1, #2, and #3) and the control mismatch siRNA are shown below.

#1 (1833-1851): (SEQ ID NO: 773) GCAGGAGCUUGUGGAAAUAUU #2 (884-902): (SEQ ID NO: 774) GCUGCAUUCUGGCAGAAAUUU #3 (357-375): (SEQ ID NO: 775) GUGCUCUGCUUAUGAUAAUUU mm (1178-1196): (SEQ ID NO: 776) CCUCGUCAUAAUACUGGGUUU

The siRNAs were expressed from the rAAV vector. Thus, to generate the siRNA #1, the DNA sequence of the sense strand GCAGGAGCTTGTGGAAATATT (SEQ ID NO:777) was linked to a spacer derived from an miRNA (TTCAAGAGA; SEQ ID NO:766) at the 3′ end, which was then linked to the corresponding antisense sequence (AATATTTCCACAAGCTCCTGC (SEQ ID NO:778)). Thus, the non-template strand sequence used in the expression cassette for the GCAGGAGCTTGTGGAAATATT (SEQ ID NO:777) siRNA will have the following sequence (SEQ ID NO:779)): GCAGGAGCTTGTGGAAATATT-TTCAAGAGA-AATATTTCCACAAGCTCCTGC. This sequence was placed downstream of an RNA polymerase III (RNA pol III) promoter in the rAAV vector. The vector also contained 6 consecutive dTs in the non-template strand following the SEQ ID NO:779 sequence to stop the transcription.

To generate the siRNA #2, the 3′ end of the DNA sequence of the sense strand GCTGCATTCTGGCAGAAATTT (SEQ ID NO:780) was linked to the TTCAAGAGA (SEQ ID NO:766) spacer which was then linked to the corresponding antisense sequence AAATTTCTGCCAGAATGCAGC (SEQ ID NO:781) to form the following shRNA sequence (SEQ ID NO:782): GCTGCATTCTGGCAGAAATTT-TTCAAGAGA-AAATTTCTGCCAGAATGCAGC.

To generate the siRNA #3, the 3′ end of the DNA sequence of the sense strand GTGCTCTGCTTATGATAATTT (SEQ ID NO:783) was linked to the TTCAAGAGA (SEQ ID NO:766) spacer which was then linked to the corresponding antisense sequence AAATTATCATAAGCAGAGCAC (SEQ ID NO:784) to form the following shRNA sequence (SEQ ID NO:785): GTGCTCTGCTTATGATAATTT-TTCAAGAGA-AAATTATCATAAGCAGAGCAC.

Note that other spacer sequences can be used instead of the TTCAAGAGA (SEQ ID NO:766) spacer. For example, the following spacer can be used: CTTCCTGTCA (SEQ ID NO:786)

As shown in FIG. 11A, each of the human ERK2 siRNAs significantly reduced ERK2 expression without significantly changing ERK1 expression, as detected by Western blot analysis of ERK1 and ERK2 protein levels in the transfected cells.

FIG. 11B graphically illustrates the reduction of ERK2 expression by each of the human ERK2 siRNAs. As shown, each of human ERK2 siRNAs #1 (SEQ ID NO:773), #2 (SEQ ID NO:774) and #3 (SEQ ID NO:775) reduced human ERK2 expression by about 70-80%.

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All patents and publications referenced or mentioned herein are indicative of the levels of skill of those skilled in the art to which the invention pertains, and each such referenced patent or publication is hereby incorporated by reference to the same extent as if it had been incorporated by reference in its entirety individually or set forth herein in its entirety. Applicants reserve the right to physically incorporate into this specification any and all materials and information from any such cited patents or publications.

The specific methods and compositions described herein are representative of preferred embodiments and are exemplary and not intended as limitations on the scope of the invention. Other objects, aspects, and embodiments will occur to those skilled in the art upon consideration of this specification, and are encompassed within the spirit of the invention as defined by the scope of the claims. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, or limitation or limitations, which is not specifically disclosed herein as essential. The methods and processes illustratively described herein suitably may be practiced in differing orders of steps, and that they are not necessarily restricted to the orders of steps indicated herein or in the claims. As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to “an antibody” includes a plurality (for example, a solution of antibodies or a series of antibody preparations) of such antibodies, and so forth. Under no circumstances may the patent be interpreted to be limited to the specific examples or embodiments or methods specifically disclosed herein. Under no circumstances may the patent be interpreted to be limited by any statement made by any Examiner or any other official or employee of the Patent and Trademark Office unless such statement is specifically and without qualification or reservation expressly adopted in a responsive writing by Applicants.

The terms and expressions that have been employed are used as terms of description and not of limitation, and there is no intent in the use of such terms and expressions to exclude any equivalent of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention as claimed. Thus, it will be understood that although the present invention has been specifically disclosed by preferred embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention as defined by the appended claims.

The invention has been described broadly and generically herein. Each of the narrower species and subgeneric groupings falling within the generic disclosure also form part of the invention. This includes the generic description of the invention with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

Other embodiments are within the following claims. In addition, where features or aspects of the invention are described in terms of Markush groups, those skilled in the art will recognize that the invention is also thereby described in terms of any individual member or subgroup of members of the Markush group. 

What is claimed is:
 1. An expression cassette comprising a promoter and a polynucleotide segment comprising a DNA or RNA corresponding to any of SEQ ID NOs: 773-775, or a combination thereof.
 2. The expression cassette claim 1, wherein the segment comprises X-L-Y, wherein X is a sense sequence corresponding to any of SEQ NOs: 773-775; L is a spacer linked to the 3′ end of the sense sequence; and Y is an antisense sequence corresponding to any SEQ ID NOs: 773-775, wherein the antisense sequence is linked to the 3′ end of the linker; and wherein the Y antisense sequence is complementary to the X sense sequence so that upon expression of the polynucleotide segment, a short hairpin RNA (shRNA) is generated.
 3. An expression vector comprising the expression cassette of claim
 1. 4. The expression vector of claim 3 wherein the vector is a viral vector.
 5. The expression vector of claim 4, wherein the viral vector is a neurotropic adeno-associated viral vector.
 6. The expression vector of claim 4, wherein the viral vector is a neurotropic recombinant adeno-associated virus (rAAV).
 7. A composition comprising a carrier and the expression cassette of claim
 1. 